Research Article: Proof of principle: Physiological transfer of small numbers of bacteria from mother to fetus in late-gestation pregnant sheep

Date Published: June 6, 2019

Publisher: Public Library of Science

Author(s): Kevin Yu, Michelle D. Rodriguez, Zubin Paul, Elizabeth Gordon, Kelly Rice, Eric W. Triplett, Maureen Keller-Wood, Charles E. Wood, Kristin Mühldorfer.

http://doi.org/10.1371/journal.pone.0217211

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. The present experiments were designed to test the hypothesis that small numbers of bacteria introduced into the maternal bloodstream (too few to induce fever or changes in maternal food consumption), can be found in the fetus days later. We injected 100 colony forming units of green-, red- and far red- fluorescent protein (GFP, RFP, FRFP) expressing S. aureus into late-gestation pregnant sheep intravenously. Five to 7 days later, the animals were euthanized and tissues collected for analysis of GFP. The inoculations did not cause any fever or other measurable behavioral response in the ewes, but did result in the appearance of GFP DNA, and protein in various tissues within the fetuses. Immunohistochemical analysis reveals GFP protein-containing bacteria that appear to be mostly contained within other cells. We were unable to recover any live GFP-expressing bacteria from the fetal tissues. We conclude that S. aureus, and perhaps other bacteria, gain access to the fetus, although it is not clear from these experiments that they survive in the fetus. It is possible that these low inocula and their progeny were effectively cleared by the fetal immune system.

Partial Text

The fetus is thought to develop in a sterile environment [1]. The presence of bacteria within the feto-placental unit is thought to represent infection–associated with a risk of premature labor [2, 3]. In recent experiments, we have discovered that transient hypoxia in pregnant sheep caused an influx of small numbers of bacteria from mother to fetus [4]; associated with that influx was a transcriptomics signature of increased activity of inflammation pathways in fetal brain [5] and kidney [6]. While the presence of bacterial DNA [7] and histological evidence of bacteria [8, 9] in human placenta has been reported, the concept that there could be live bacteria transferred to the fetal compartment is debated [10]. In our own experiments [4], we used whole genome sequencing to prove live bacteria isolated from the placenta and fetal brain after hypoxia were identical, suggesting that there is transfer of bacteria from placenta to the fetus under the right conditions. While our data strongly suggest the transfer of live bacteria from mother to fetus, our previous experiments did not provide proof of this concept. We designed the present study to directly test the hypothesis that bacteria in maternal blood can cross the maternal/fetal barrier and into the fetus.

We have confirmed our hypothesis that S. aureus introduced into the maternal bloodstream crosses the maternal/fetal barrier and enters into the fetus. While we were able to demonstrate the appearance of GFP DNA and protein in the fetus, we were unable to demonstrate that the GFP-containing bacteria in the fetus were alive. Our inability to find live GFP-expressing bacteria in the fetus might be the result of the time interval allowed between inoculation and tissue collection (6 days). The design of the experiment does not allow us to determine whether the bacteria were killed upon transit from mother to fetus or whether the bacteria entered the fetus alive, then were killed by the fetal immune system. Consistent with the absence of detectable live bacteria is the appearance of the GFP protein in the tissue, which appeared as aggregated clumps rather than with morphology consistent with intact live bacteria. The appearance of bacteria in aggregated clumps as well as their low abundance in the fetal tissues may explain the discordance amongst labels (GFP, RFP, FRFP) in detection of plasmid DNA in fetal livers. The DNA extraction protocol uses approximately 100-150mg of tissue and RFP bacteria may have been absent in those specific tissue pieces used for extraction.

 

Source:

http://doi.org/10.1371/journal.pone.0217211

 

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