Development of the preterm infant gut and gastric residuals microbiome.

Prematurity, defined as birth before 37 weeks of gestation, is the leading cause of mortality in children under five, affecting ~11% of live births worldwide (≈15 million annually). Despite advances in neonatal care, preterm infants remain at high risk of complications. In neonatal intensive care units, gastric residuals (GRs) are routinely monitored to guide enteral feeding, yet their microbial composition remains poorly understood. We performed metagenomic sequencing of 199 stool and 69 GR samples from 39 preterm infants during hospitalization to characterize stomach and gut microbiomes. To our knowledge, this is the first metagenomic sequencing of the GR in premature infants. We identified 11 GR microbial clusters, commonly dominated by Staphylococcus, Streptococcus, and Klebsiella, with microbial diversity correlating with aspiration frequency. Colonization was dynamic: early GR samples were enriched with Staphylococcus epidermidis and Bradyrhizobium, while later samples featured Escherichia coli, Staphylococcus hominis, and Streptococcus thermophilus. Stool samples formed eight microbial clusters, frequently enriched with Enterobacteriaceae. S. epidermidis was linked to higher gestational age and lower richness, whereas Bifidobacterium breve, a beneficial commensal, appeared later. Comparative analysis showed overlap between gut and gastric microbiota, with GR samples more dynamic and less subject-specific. Strain-level analysis revealed both individual-specific and widely shared taxa, including a pathogenic Klebsiella aerogenes strain associated with bacteremia, detectable a week before clinical isolation. These findings provide new insights into microbial colonization dynamics of preterm infants.