BACKGROUND: Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth but the role of the microbiome in its development remains unclear. We, therefore, assessed the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways, and assessed the gut-lung axis by comparing the upper and lower airways bacterial communities with the stool findings. Finally, we assessed if the bacterial communities were associated with lung inflammation to suggest dysbiosis. METHODS: We serially sampled multiple anatomical sites including the upper airway (nasopharyngeal aspirates, NPA), lower airways (tracheal aspirate fluid, TAF, and bronchoalveolar lavage fluid, BAL) and the gut (stool) of ventilated preterm-born infants. Bacterial DNA load was measured in all samples and sequenced using the V3-V4 region of the 16S rRNA gene RESULTS: From 1102 (539 NPA, 276 TAF, 89 BAL, 198 stool) samples from 55 preterm infants, 352 (32%) amplified suitably for 16 s RNA gene sequencing. Bacterial load was low at birth, quickly increased with time but was associated with predominant operational taxonomic units (OTUs) in all sample types. There was dissimilarity in bacterial communities between the upper and lower airways and the gut with a separate dysbiotic inflammatory process occurring in the lower airways of infants. Individual OTUs were associated with increased inflammatory markers. CONCLUSIONS: Taken together, these findings suggest that targeted treatment of the predominant organisms, including those not routinely treated such as Ureaplasma spp., may decrease the development of CLD in preterm-born infants.