The fetus is thought to develop in a sterile environment in utero. Long standing dogma that “the uterus and the feto‐placental unit is “sterile” is based primarily on microbiological culture‐based techniques that were unsuccessful in growing “culture resistant” bacteria or intracellular bacteria. We have reported the presence of low numbers of bacteria in tissues from normal sheep fetuses in pregnancies not complicated by infection. The exposure of the fetus to bacteria might aid neonatal survival by informing fetal immune development. We propose that the fetus is not sterile and that bacteria or fragments of bacteria can be transferred from mother to fetus. We hypothesize that inoculation in the maternal mouth results in the appearance of bacteria in the fetus. We used S. aureus containing green (GFP), red (RFP), or far‐red (FRFP) fluorescent protein‐expressing plasmids to inoculate late‐gestation pregnant sheep (gestational age= 130–135 days, n=7) intravenously (GFP, 104 cfu), into maternal mouth (RFP, 104 cfu) and vagina (FRFP, 104 cfu). These were small doses of bacteria which did not cause physiological (no fever) or psychological (no anorexia) signs of infection. Five to seven days post maternal inoculation, animals were humanely sacrificed, and fetal tissues were collected, and DNA was extracted from placenta and fetal liver, spleen, and cerebral cortex. We probed for GFP plasmid using several primer pairs for endpoint PCR. While detection of whole‐length plasmids was not successful, PCR reactions probing for smaller fragments of plasmid were successful. We found GFP plasmid DNA in all tissues in 10/10 fetal livers and RFP in 10/10 livers. The appearance of GFP and RFP‐labelled bacteria in fetal liver (p=7.497e‐06) was statistically significant as tested by Chi‐Square analysis. We did not detect significant FRFP plasmid DNA in liver. Analysis of tissues by immunohistochemistry revealed GFP and RFP‐expressing bacteria in fetal tissues, although most appeared to be in aggregates. We conclude that S. aureus, introduced in small numbers in maternal mouth and bloodstream, appear in the fetus and placenta. We were unable to determine whether these bacteria were alive in the fetus. Support or Funding Information This work was supported by HD033053, AI120195, and HL083810 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources.

The fetus develops in a privileged environment, as the placenta serves as both a gateway for nutrients and a barrier for pathogen transfer to the fetus. Regardless, recent evidence suggests the presence of bacterial DNA in both placenta and fetus, and we have reported that DNA and protein from small numbers of bacteria gain access to the fetus from the maternal bloodstream. Other routes of environmental bacterial transfer from the mother to fetus remain unknown, as well as the physiological relevance of their presence. In these experiments, we examine multiple routes by which bacterial cellular components can enter the fetus and the fetal response to influx of bacterial DNA and protein. We inoculated maternal sheep with genetically-labeled S. aureus (Staphylococcus aureus) using three routes: intravenously, orally, and intra-vaginally. The inoculum did not produce sepsis or fever in the ewes, therefore mimicking incidental exposure to bacteria during pregnancy. 3–5 days post inoculation, we assessed the presence of bacterial components in the fetal tissues and analyzed fetal brain tissue to identify any alterations in gene expression. Our results demonstrate that components of bacteria that were introduced into the maternal mouth were detected in the fetal brain and that they stimulated changes in gene expression. We conclude that an oral route of transmission is relevant for transfer of bacterial cellular components to the fetus.