Meconium constitutes infants' first bowel movements postnatally. The consistency and microbial load of meconium are different from infant and adult stool. While recent evidence suggests that meconium is sterile in utero, rapid colonization occurs after birth. The meconium microbiome has been associated with negative health outcomes, but its composition is not well described, especially in preterm infants. Here, we characterized the meconium microbiomes from 330 very preterm infants (gestational ages 28 to 32 weeks) from 15 hospitals in Germany and in fecal samples from a subset of their mothers (N = 217). Microbiome profiles were compiled using 16S rRNA gene sequencing with negative and positive controls. The meconium microbiome was dominated by Bifidobacterium, Staphylococcus, and Enterococcus spp. and was associated with gestational age at birth and age at sample collection. Bifidobacterial abundance was negatively correlated with potentially pathogenic genera. The amount of bacterial DNA in meconium samples varied greatly across samples and was associated with the time since birth but not with gestational age or hospital site. In samples with low bacterial load, human mitochondrial sequences were highly amplified using commonly used, bacterial-targeted 16S rRNA primers. Only half of the meconium samples contained sufficient bacterial material to study the microbiome using a standard approach. To facilitate future meconium studies, we present a five-level scoring system ("MecBac") that predicts the success of 16S rRNA bacterial sequencing for meconium samples. These findings provide a foundational characterization of an understudied portion of the human microbiome and will aid the design of future meconium microbiome studies. IMPORTANCE Meconium is present in the intestines of infants before and after birth and constitutes their first bowel movements postnatally. The consistency, composition and microbial load of meconium is largely different from infant and adult stool. While recent evidence suggests that meconium is sterile in utero, rapid colonization occurs after birth. The meconium microbiome has been associated with short-term and long-term negative health outcomes, but its composition is not yet well described, especially in preterm infants. We provide a characterization of the microbiome structure and composition of infant meconium and maternal feces from a large study cohort and propose a method to evaluate meconium samples for bacterial sequencing suitability. These findings provide a foundational characterization of an understudied portion of the human microbiome and will aid the design of future meconium microbiome studies.

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