Preterm neonates are increasingly being researched for microbial ecosystems that influence health and illness. However, there is limited evidence on longitudinal microbial acquisition, particularly in the most premature infants. The goal of this research is to describe the development of the microbiota in this hitherto under-represented population. A single neonatal critical care unit was used to enrol seven severely preterm infant-mother dyads. Over the first 60 days of life, oral and endotracheal secretions, faeces, and breast milk were collected. Bacterial communities were discovered using targeted 16S rRNA gene sequencing. The microbiota of all body locations were most comparable shortly after birth and thereafter differed longitudinally. Throughout the sample period, Escherichia, Enterococcus, Staphylococcus, and Enterobacteriaceae were prevalent and widely distributed. The decreased diversity and considerably higher relative abundance of Bifidobacteriaceae in the stool compared to other locations drove the stool’s temporal separation from other microbiota.

All anatomical sample locations were dominated by four taxa. Dissimilarity was encouraged by rare taxa. Cross-seeding was seen between upstream populations and the stool, which might be related to buccal colostrum/breast milk exposure and indwelling tubes.Given the significance of dysbiosis in the health and illness of extremely preterm babies, a greater knowledge of microbial acquisition in this environment might be clinically beneficial.