Early Neonatal Meconium Does Not Have a Demonstrable Microbiota Determined through Use of Robust Negative Controls with cpn60-Based Microbiome Profiling

Abstract

ABSTRACT Detection of bacterial DNA within meconium is often cited as evidence supporting in utero colonization. However, many studies fail to adequately control for contamination. We aimed to define the microbial content of meconium under properly controlled conditions. DNA was extracted from 141 meconium samples and subjected to cpn60-based microbiome profiling, with controls to assess contamination throughout. Total bacterial loads of neonatal meconium, infant stool, and controls were compared by 16S rRNA quantitative PCR (qPCR). Viable bacteria within meconium were cultured, and isolate clonality was assessed by pulsed-field gel electrophoresis (PFGE). Meconium samples did not differ significantly from controls with respect to read numbers or taxonomic composition. Twenty (14%) outliers with markedly higher read numbers were collected significantly later after birth and appeared more like transitional stool than meconium. Total bacterial loads were significantly higher in stool than in meconium, which did not differ from that of sequencing controls, and correlated well with read numbers. Cultured isolates were most frequently identified as Staphylococcus epidermidis, Enterococcus faecalis, or Escherichia coli, with PFGE indicating high intraspecies diversity. Our findings highlight the importance of robust controls in studies of low microbial biomass samples and argue against meaningful bacterial colonization in utero. Given that meconium microbiome profiles could not be distinguished from sequencing controls, and that viable bacteria within meconium appeared uncommon and largely consistent with postnatal skin colonization, there does not appear to be a meconium microbiota. IMPORTANCE Much like the recent placental microbiome controversy, studies of neonatal meconium reporting bacterial communities within the fetal and neonatal gut imply that microbial colonization begins prior to birth. However, recent work has shown that placental microbiomes almost exclusively represent contamination from lab reagents and the environment. Here, we demonstrate that prior studies of neonatal meconium are impacted by the same issue, showing that the microbial content of meconium does not differ from negative controls that have never contained any biological material. Our culture findings similarly supported this notion and largely comprised bacteria normally associated with healthy skin. Overall, our work adds to the growing body of evidence against the in utero colonization hypothesis.

Keywords

• contamination
• cpn60
• low biomass
• meconium
• microbiome
• negative controls