The human body is home to a dynamic and evolving community of microorganisms that collectively form the microbiome. The first months of life are critical for the acquisition of the microbiome and mark the earliest interactions between our microbiota and maturing immune system. The lung, previously believed to be sterile, harbours a variety of microorganisms recently thought to be critical in these processes. Although metagenomic technologies continue to uncover the diverse microbial community in the human respiratory tract, isolate-level resolution, dynamic developmental changes, and functional analysis of the microbiota across body sites is yet to be fully achieved. Through the ‘GLAM&I’ (Gut and Lung and their Microbiome and Immunology) clinical study, longitudinal and time-matched bronchoalveolar lavage, nasopharyngeal swab, stool, blood, and milk samples have been sampled from 79 patients admitted to the neonatal and paediatric intensive care units at Monash Children’s Hospital. To evaluate community composition, we have metagenomically sampled respiratory and stool microbial communities and cultured and purified individual bacterial isolates to allow functional validation of the interactions between the microbiota and host immune system. Capillary sequencing of the 16S rRNA gene in 5,480 individual isolates from 208 respiratory samples and 1,657 isolates from 62 stool samples has revealed 30 bacterial species shared across the respiratory tract and gut microbiomes in early life. Future work will validate the site-specific functional roles and immune interaction of these isolates. The application of culturing to the lung microbiome will improve our understanding of host-microbe interactions and support experimental validation of patient-specific bacterial species. This knowledge has the potential to provide novel therapeutics and microbiome-based medicines.