Abstract
The human microbiome includes trillions of bacteria, many of which play a vital role in host physiology. Numerous studies have now detected bacterial DNA in first-pass meconium and amniotic fluid samples, suggesting that the human microbiome may commence in utero. However, these data have remained contentious due to underlying contamination issues. Here, we have used a previously described method for reducing contamination in microbiome workflows to determine if there is a fetal bacterial microbiome beyond the level of background contamination. We recruited 50 women undergoing non-emergency cesarean section deliveries with no evidence of intra-uterine infection and collected first-pass meconium and amniotic fluid samples. Full-length 16S rRNA gene sequencing was performed using PacBio SMRT cell technology, to allow high resolution profiling of the fetal gut and amniotic fluid bacterial microbiomes. Levels of inflammatory cytokines were measured in amniotic fluid, and levels of immunomodulatory short chain fatty acids (SCFAs) were quantified in meconium. All meconium samples and most amniotic fluid samples (36/43) contained bacterial DNA. The meconium microbiome was dominated by reads that mapped to Pelomonas puraquae. Aside from this species, the meconium microbiome was remarkably heterogeneous between patients. The amniotic fluid microbiome was more diverse and contained mainly reads that mapped to typical skin commensals, including Propionibacterium acnes and Staphylococcus spp. All meconium samples contained acetate and propionate, at ratios similar to those previously reported in infants. P. puraquae reads were inversely correlated with meconium propionate levels. Amniotic fluid cytokine levels were associated with the amniotic fluid microbiome. Our results demonstrate that bacterial DNA and SCFAs are present in utero, and have the potential to influence the developing fetal immune system.
Highlights
It has long been assumed that the human fetus is sterile
Our data suggest that the fetus is exposed to bacterial DNA and metabolites prior to birth
Additional studies that utilize pre-extraction sample treatment with membrane-impermeable dyes such as propidium monoazide to prevent PCR amplification of DNA contained within compromised cell walls will help to shed light on whether this DNA originates from viable or dead bacteria and as such whether it constitutes a true microbiome
Summary
It has long been assumed that the human fetus is sterile. Over the last decade new data have emerged to challenge this dogma. It is well accepted that laboratory reagents, including nucleic acid extraction kits and PCR master mix reagents, harbor lows levels of bacterial DNA (Salter et al, 2014) While this contamination is not a major issue for studies of highly colonized samples such as adult feces, it becomes an important issue when working with low biomass samples such as the meconium and amniotic fluid. Issues around contamination and interpretation of low-biomass data remain, with several studies finding that the placental “microbiome” is indistinguishable from negative controls (Lauder et al, 2016; Hornef and Penders, 2017; Leiby et al, 2018; Theis et al, 2019) This is a significant consideration as bacteria entering the intra-amniotic space would necessarily pass through the placenta or the extra-placental membranes
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