The Composition of the Microbiota in the Full-Term Fetal Gut and Amniotic Fluid: A Bovine Cesarean Section Study

Abstract

The development of a healthy intestinal immune system requires early microbial exposure. However, it remains unclear whether microbial exposure already begins at the prenatal stage. Analysis of such low microbial biomass environments are challenging due to contamination issues. The aims of the current study were to assess the bacterial load and characterize the bacterial composition of the amniotic fluid and meconium of full-term calves, leading to a better knowledge of prenatal bacterial seeding of the fetal intestine. Amniotic fluid and rectal meconium samples were collected during and immediately after elective cesarean section, performed in 25 Belgian Blue cow-calf couples. The samples were analyzed by qPCR, bacterial culture using GAM agar and 16S rRNA gene amplicon sequencing. To minimize the effects of contaminants, we included multiple technical controls and stringently filtered the 16S rRNA gene sequencing data to exclude putative contaminant sequences. The meconium samples contained a significantly higher amount of bacterial DNA than the negative controls and 5 of 24 samples contained culturable bacteria. In the amniotic fluid, the amount of bacterial DNA was not significantly different from the negative controls and all samples were culture negative. Bacterial sequences were identified in both sample types and were primarily of phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with some individual variation. We conclude that most calves encounter in utero maternal-fetal transmission of bacterial DNA, but the amount of bacterial DNA is low and viable bacteria are rare.

Characterization of the microbiota associated with 12-week-old bovine fetuses exposed to divergent in utero nutrition

“…The fetal bacterial microbiota was dominated by the Proteobacteria, Firmicutes, Actinobacteriota and Bacteroidota phyla. Similar to our study, previous studies reported the presence of an archaeal and bacterial microbiota in amniotic fluid and the intestine of 5-, 6-and 7month-old fetuses (Angus × Friesian bred) (Guzman et al, 2020), and C-section derived near fullterm calf fetuses (Belgian Blue) (Husso et al, 2021). The dominant bacterial phyla observed in the 83-day-old fetuses in the present study were also the predominant phyla in these mid-and fullterm calf fetuses and in the calves postnatally.…”

“…However, this was challenged by recent evidence showing that prenatal microbial colonization may take place in the intestine of fetal calves. Results of a bovine caesarean section study that was conducted to evaluate bacterial load and the bacterial composition of the amniotic fluid and meconium of near full-term calves using 16S rRNA gene sequencing, qPCR and culturing suggested that in utero maternal-fetal bacterial transmission may occur before birth in calves (Husso et al, 2021). In addition, Guzman et al, (2020) investigated the presence of a microbiota in amniotic fluid and in 5 sample types from the gastrointestinal tract (GIT) of 5-, 6-and 7-monthold calf fetuses using both molecular and culture-based approaches.…”

From Short- to Long-Term Effects of C-Section Delivery on Microbiome Establishment and Host Health

“…In another study, bacteria were found in fetal membranes and umbilical cords but never inside the placenta, supporting the hypothesis of contamination as the source of bacterial presence in studies that challenge the "sterile womb" hypothesis [15]. As for the amniotic fluid, a recent study in a bovine C-section model revealed some positives (5 out of 24), with low amounts of bacteria in the meconium, but none of the amniotic fluid samples presented different amounts of bacterial DNA compared to the negative controls [16]. In a recent study, fecal meconium was collected before antibiotic administration during breech CSDs without labor and compared to standard VDs, first-pass meconium, and stool.…”

Administration of All-Trans Retinoic Acid to Pregnant Sows Alters Gut Bacterial Community of Neonatal Piglets With Different Hoxa1 Genotypes

“…Maternal genetics ( Goodrich et al, 2014 ), gene mutation ( Strati et al, 2016 ), delivery methods ( Bi et al, 2021 ; Husso et al, 2021 ), age ( Yatsunenko et al, 2012 ), disease ( Alkanani et al, 2015 ), diet ( David et al, 2014 ; Angoa-Pérez et al, 2020 ), and medication ( Forslund et al, 2013 ) are the dominant factors in shaping the composition and abundance of intestinal microbiota. It is reported that the composition of intestinal bacterial communities in human and other mammals after suckling is mainly dominated by the phyla Firmicutes and Bacteroidetes, followed by the phyla Proteobacteria, Actinobacteria, and Verrucomicrobia ( Gill et al, 2006 ; Duncan et al, 2008 ; Ley et al, 2008 ); however, the compositions of bacteria in the meconium of fetal bovine and lamb delivered by cesarean section were primarily composed of the phyla Proteobacteria and Firmicutes instead of Firmicutes and Bacteroidetes ( Bi et al, 2021 ; Husso et al, 2021 ), and data in our study also indicated that Firmicutes and Proteobacteria were the most abundant phyla in the meconium of vaginal-delivered neonatal piglets before suckling.…”

“…Gut microbiota is integral to feed digestion, nutrient absorption and metabolism, immune response, and gastrointestinal development ( Morgavi et al, 2015 ), and the colonization of intestinal microbiota during early life could further influence the subsequent microbiota of adult host ( Ben Salem et al, 2005 ). Many studies demonstrated that the intestine of prenatal animals really has microorganism ( Alipour et al, 2018 ; Stinson et al, 2019 ; Hummel et al, 2020 ; Bi et al, 2021 ; Husso et al, 2021 ), and at present, no literature on the differences in intestinal microbiota composition between mutant and wild-type fetuses is found, but for postnatal individuals, there are differences in gut microbiota between mutant and wild-type host, for example, nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutation caused Crohn’s disease (CD) ( Hampe et al, 2001 ; Ogura et al, 2001 ) and Crohn’s disease individuals had lower bacterial diversity than healthy controls ( Joossens et al, 2011 ). Cystic fibrosis transmembrane conductance regulator (CFTR) mutation resulted in multiorgan defects, and CFTR –/– mice had significantly lower alpha diversity of intestinal bacterial community ( p < 0.05) and had reduced relative abundance of protective species such as Acinetobacter lwoffii and Lactobacilliales members compared with wild-type mice ( Lynch et al, 2013 ).…”