Fetal exposure to the maternal microbiota in humans and mice

Abstract

Previous studies have demonstrated the presence of microbial DNA in the fetal environment. However, it remains unclear whether this DNA represents viable bacteria and how it relates to the maternal microbiota across body sites. We studied the microbiota of human and mouse dyads to understand these relationships, localize bacteria in the fetus, and demonstrate bacterial viability. In human preterm and full-term mother-infant dyads at the time of cesarean delivery, the oral cavity and meconium of newborn infants born as early as 24 weeks of gestation contained a microbiota that was predicted to originate from in utero sources, including the placenta. Using operative deliveries of pregnant mice under highly controlled, sterile conditions in the laboratory, composition, visualization, and viability of bacteria in the in utero compartment and fetal intestine were demonstrated by 16S rRNA gene sequencing, fluorescence in situ hybridization, and bacterial culture. The composition and predicted source of the fetal gut microbiota shifted between mid- and late gestation. Cultivatable bacteria in the fetal intestine were found during mid-gestation but not late gestation. Our results demonstrate a dynamic, viable mammalian fetal microbiota during in utero development.

Keywords: Bacterial infections; Development; Microbiology.

Figure 1. Sequencing-based determination of the microbiota of human preterm and full-term mother-infant dyads delivered by cesarean delivery.

(A) Principal coordinates analysis of samples based on Bray-Curtis distances. (B) Pairwise comparisons of Bray-Curtis distances between sample sites by adonis PERMANOVA with Benjamini-Hochberg adjustment. The R² value and P value (in parentheses; highlighted if P < 0.05) are noted for each comparison. (C) Heatmap of the relative proportion of the top bacterial taxa (lowest assigned taxonomy) represented within each sample site in premature and full-term mother-infant pairs.

Figure 2. Proportions of the infant microbiota attributed to maternal and in utero sources.

Predicted sources of the infant microbiota were determined using SourceTracker (26). The proportion of infant microbiota attributed to the maternal and in utero sources are shown for the infant’s oral cavity and feces (i.e., meconium) among preterm (A) and full-term (B) mother-infant dyads. The subjects did not experience labor or rupture of membranes before the delivery, with the exceptions of subject PT3 (preterm labor and rupture of membranes) and subject PT5 (preterm labor without rupture of membranes). The preterm infants PT1, PT2, PT3, and PT5 received antibiotics after birth.

Figure 3. Relative abundance of top bacterial taxa in murine maternal and fetal microbiota across gestational stages.

The top 15 bacterial taxa (lowest assigned taxonomy) within each maternal or fetal site is shown among early-mid (E14–16), mid-late (E17–18), or late (E19–20) gestation mice. Samples from 2 dams and 3–5 fetuses per dam are represented.

Figure 4. Identification of bacterial DNA in the murine fetal intestine by FISH of the 16S rRNA gene.

Intestinal tissue sections were obtained from fetal mice at E12–14 and were stained with DAPI and probed with Cy3-labeled eubacterial probe EUB338 and a scrambled-probe control. Images were acquired by confocal microscopy. Scale bar: 50 μm. Magnification, 10×.

Figure 5. Predicted sources of the murine fetal microbiota.

Bacterial source tracking was used to determine the likely sources of bacterial DNA in the gut and skin of early-mid (E14–16), mid-late (E17–18), and late (E19–20) gestation fetuses. Samples from 2 dams and their corresponding fetuses are represented.

Figure 6. Bacterial strains cultured from the murine fetal intestine, placenta, and uterus.

The top gray-and-white bar indicates individual dams and the embryonic day at the time of sampling, followed by the anatomic site from which the bacterial strains were isolated. Colored squares in the heatmap represent the sites with positive cultures. Fewer samples had positive cultures in late gestation. Strains were identified by Sanger sequencing of the 16S rRNA gene (8F/1492R). The accession number for the top match identified by the Basic Local Alignment Search Tool (BLAST) is shown for each isolate (isolates without a match of at least 94% sequence identity were designated as unknown).

Figure 7. Cultivation of streptomycin-resistant E. coli from maternal and fetal tissues following maternal colonization.

(A) Timeline for treatment of dams with antibiotics (Abx; streptomycin), colonization with streptomycin-resistant E. coli by oral gavage, and collection of maternal and fetal tissues. (B) Colony-forming units (CFU) counts per plate (100 μl homogenate/plate) from the maternal and fetal tissues in early gestation (E9). (C) CFU counts per plate (100 μl homogenate/plate) from the maternal and fetal tissues in mid-late gestation (E18). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.