We implemented 16S rRNA sequencing using the Pacbio RS system to explore the meconium microbiome of 23 newborns. Our findings showed that the meconium was not sterile in all of our study subjects and contained diverse microbiota communities, regardless of the demographic and clinical characteristics. A number of studies using culture-dependent or independent methods have established the presence of live bacteria in the fetus prior to birth 
. However, until the development of the next generation sequencing (NGS) technology, the detailed characterization of the microbiome at the taxa level was challenging. Moreover, while a number of studies have compared the bacterial composition of the adult stool with early infant stool 
, only a handful of small studies have been carried out in meconium 
. Using a culture-independent NGS technology, we demonstrated that the meconium microbiota substantially differ from those in adult feces, with the overall microbiome diversity being significantly lower than that of the adult stool (). While at the phylum level the meconium microbiome was more similar to that of infants 
than adults, at the genus level, the dominant taxa found in the early infant stool were not prevalent in the meconium samples.
Among the meconium samples, we observed a significant increase in the diversity of various bacterial types if mothers had pre-gestational DM compared to those with no diabetes or GDM.
These results are also consistent with the recent findings that have suggested that certain microbiota components are more prevalent in adult individuals with DM 
. Specifically, at the OTU level, the Bacteroides
, which have been reportedly prevalent in adult diabetes patients 
, were enriched in the meconium of babies born to mothers with DM. Despite the enrichment of certain bacteria, a previous study have reported no difference in the mean OTU diversity within each sample between the adult DM cases and controls 
. However, in our study, we observed that the meconium in the DM group showed higher alpha-diversity than that in the no-diabetes or GDM groups with a possible explanation being that the bacterial transmission to fetus may be more permissible under maternal diabetic conditions.
An additional illustration of the link between the meconium microbiota composition and maternal and infant health has been provided by Gosalbes et al
., who reported that the meconium microbiota types dominated by lactic acid or enteric bacteria are differentially associated with maternal eczema and respiratory problems in infants 
. Moreover, they have shown that some of the species that reach the fetal gastrointestinal tract prenatally can be present in the infant long into the first year, underlining the potential long-term clinical consequences of the initial microbiota content. We speculate that the bacterial diversity in the gut of a fetus may reflect pathophysiological processes occurring during pregnancy or represent a direct transmission of maternal intestinal bacteria. Prospective studies will be required to further clarify the path of the mother-to-baby efflux of commensal microbes during pregnancy, as well as the impact of the maternal microbiome on predisposition to adult-onset diseases.
Microbiome studies of early infancy have demonstrated a significant effect of the mode of delivery on the microbiome composition, suggesting the likely association of the infant gut bacteria with maternal vaginal or skin microbiome habitats 
. However, in our study, no significant differences in the overall microbiome composition by the mode of delivery were detected in babies born to mothers with and without diabetes. Furthermore, we observed that neither of the dominant OTUs reported previously 
at the genus level in the stool of young infants delivered vaginally (Lactobacillus
) or via C-section (Acinetobacter
) were prevalent in the majority of the meconium samples. Instead, the most common OTUs at the genus level in the meconium included Comamonas
, which are predominantly aerobic or facultative anaerobic organisms. Comamonas
was found in human appendix, a small pouch attached to the first portion of the large intestine 
occur naturally in the human gut 
, further supporting our hypothesis that the initial colonization in the human gut starts prior to birth. While our findings are consistent with a recent report that also showed no substantial overlap of the meconium microbiota with maternal habitats, especially in babies born via C-section 
, it contrasts previous analyses demonstrating that the microbiota recovered from multiple newborn sites closely resembles vaginal or skin microbiota, depending on the mode of delivery 
. Emerging studies point toward pregnancy as the beginning of bacterial exposure for the developing fetus 
. A recent study on the microbiome dynamics in the gut of pregnant women has shown that the bacterial composition evolves between the 1st
trimesters, with the maternal gut microbiome in the 3rd
trimester being more similar to what we observed in the meconium, including the increased variability in beta-diversity, the compositional dissimilarity among samples, increasing abundance of Proteobacteria
, and the reduction in alpha-diversity between samples 
. Taken together, it further suggests that the maternal microbiota can be transferred to the fetus during pregnancy.
For this study, we used the Pacbio RS system. Compared with other NGS platforms, Pacbio RS has the advantage of a much shorter sequencing time (1 to 2 hours) and the much longer read length (up to 8,000 bases). However, the embedded relatively high random sequencing error rate was until recently considered a limiting factor for applying this technology to 16S-based taxonomy assignment and phylogenetic analysis. Our result showed that using CCS reads we were able to replicate an earlier experiment and correctly assign the OTUs to at least the genus level.
This study's limitations include the fact that the sample size may not have allowed us to detect modest differences in bacterial distribution. Nevertheless, it was comparable to the size of a recent study with similar objectives and design 
and allowed us to detect significant differences with regard to maternal diabetes status. Also, we did not have access to the matched maternal samples; thus, evidence connecting the fetal microbiome directly to the maternal microbiome is lacking. Future studies comparing bacterial composition of the matched maternal (vaginal, placental and fecal) and neonatal microbiome are warranted to determine the major source of the newborn microbiota. In addition, since the gut microbiota changes dramatically during pregnancy 
, a direct comparison between pregnant and non-pregnant women with DM will be important to explore the effect of pregnancy on bacterial content in the context of DM. Furthermore, numerous prenatal factors, such as chorioamnonitis or premature rupture of membranes, maternal prenatal smoking, maternal stress, and others , may contribute to the initial colonization of the gut microbiome. Women were also excluded from the study if they received antibiotic treatment over the course of pregnancy, precluding testing the relationship between maternal infection, diabetes, and meconium content. However, women undergoing C-section and receiving a prophylactic dose of an antibiotic as a standard of care were retained in the study, which could potentially affect the meconium microbiota, especially if antibiotics were given before cord clamping. The sample size of this study would not allow to systematically assess the effect of these and other factors on the meconium microbiome profile. Larger studies collecting extensive prenatal and neonatal data are underway to explore the role of other factors that have effects on the meconium microbiome. Also, health-related information, such as BMI, maternal smoking or glucose level, was not accessible on several enrolled subjects due to the lack of consent or ethical issues associated with administering GCT to individuals with established DM. Therefore, we were unable to establish the relationship between these traits and the meconium microbial composition. Moreover, the time of sampling widely varied among the newborns raising the possibility that environmental exposures may have influenced the bacterial content for those who passed their first stool at a later time. However, the time of sampling was not significantly different between the study groups; therefore, it is unlikely to systematically bias our results. Also, it is known that maternal diabetes can increase the risk of pre-term birth, due to an increase in both fetal and maternal indications for delivery. In our study, we did not see microbiota differences by gestational age among DM, GDM, and no-diabetes groups; however, future studies with a larger sample size will have to examine whether gestational age at delivery is associated with the meconium microbiota. In addition, we did not account for susceptibility loci predisposing to DM, which could also be transmitted from a mother to an infant and potentially affect the microbiome. Future studies will have to determine if host DM risk alleles are associated with a “diabetic” microbial profile.
In summary, our study provides further evidence that meconium contains diversified microbiota and suggests that the initial colonization of the gut flora may start prior to birth. Furthermore, the meconium microbiome of babies born to DM mothers is enriched for the bacterial OTUs observed in the fecal microbiome of adult DM patients. These findings can enhance our understanding of a non-genetic risk of transmission of DM, and help design novel preventive measures for adult onset diseases.