Discovery of Bacteria in Meconium of Normal, Uncatheterized, and Uninfected Late‐Gestation Fetal Sheep

Abstract

Fetal development is thought to proceed in a sterile environment. Recent reports of the presence of bacterial DNA in human placenta, the transfer of live bacteria from mother to fetus after hypoxia in the pregnant sheep, and the presence of bacteria in the meconium of newborn infants have suggested that the fetus might be exposed to bacteria in utero. We performed the present studies to build on our discovery of bacterial rRNA in the meconium of normal fetal sheep. Specifically, the present experiments were designed to test the hypothesis that intact bacteria could be visualized histologically in meconium from normal, uncatheterized, and uninfected fetal sheep. Pregnant ewes, uncatheterized and free of clinical disease, were euthanized in late gestation. At necropsy, meconium was isolated from fetal colon using RNAse and DNAse‐free instruments and snap‐frozen, then stored at −80C until analyzed. Approximately 150 mg of meconium was homogenized and both RNA and DNA extracted in Trizol. RNA was isolated, converted to cDNA using random hexamers, then subjected to endpoint PCR using primers 341F and 806R that amplify a 465 bp region of bacterial 16S rRNA. All assays were performed with both positive and negative controls. In meconium, 7/7 fetuses (143 days gestation; term=147 days) harbored bacterial RNA. Smears of meconium were stained using Gram stain or hematoxylin stain. Gram stain revealed Gram positive and Gram negative organisms with morphologies that are consistent with the genus/species of bacteria that we have previously identified in meconium using 16S rRNA gene sequencing in DNA isolated from meconium. Hematoxylin staining confirmed the morphology of bacteria observed in the Gram‐stained samples. Bacteria in meconium appear in clusters that are consistent with live, replicating, organisms. The present results support previous work from this laboratory demonstrating that bacteria gain access to the normal fetal sheep. These results are consistent with our observations that bacteria are present in low numbers in amniotic fluid. It is possible that amniotic fluid containing bacteria is swallowed by the fetus, that bacteria survive the transit through the immature fetal gastrointestinal tract, and that the bacterial population expands in the nutrient‐rich environment of the fetal colon.Support or Funding InformationThis work was supported by HD033053 and AI120195.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.