PROJECT SUMMARY Infancy is increasingly being recognized as a key time point of microbiome establishment that impacts neonatal health as well as later outcomes. The intestinal microbiome has specifically been implicated in neurologic outcomes via the gut-brain axis. However, means by which the intestinal microbiome can have influence on the brain are poorly understood. The preterm infant is at the nexus of these unknowns. Preterm infants are a vulnerable patient population at risk for significant poor long-term neurodevelopmental outcomes. Preterm infant brain development occurs in parallel with intestinal microbiome development, thus modification of the intestinal microbiome is a potential means of improving neurodevelopmental outcomes. In this proposal, we will test the hypothesis that distinct gut microbiome taxa and metabolites at key time points improve preterm infant neurodevelopmental outcomes at school age. Our preliminary and published data in gnotobiotic mouse models demonstrates that different early preterm infant microbiota impact neuron number, myelination, and behavior. This proposal will use our ongoing MIND (Microbiome In Neonatal Development) preterm infant cohort to determine how the gut microbiome impacts neurodevelopmental potential in the NICU, and how it may alter neurodevelopmental trajectories post- NICU discharge. We will conduct longitudinal sampling of participant fecal and blood samples to monitor gut microbiome as well as fecal and serum metabolites. We will also perform neurodevelopmental testing during the NICU course and up until preschool/school age (3.5-5 years old). School readiness, which describes children's strengths, challenges, and needs for supports when learning in the classroom, is a functional outcome that differs from single summary measures of intelligence (IQ) and will be the outcome measure. A combination of 16S rRNA gene sequencing, metagenomics and metabonomics will be applied to the collected fecal samples. Sophisticated machine learning strategies will be used to develop novel models of preterm infant gut microbiome succession with time as a critical element. Serum cytokine analysis and metabonomics will provide mechanistic insight into how the gut microbiome may be impacting neurodevelopment. We have established complementary in vivo gnotobiotic mouse models, in which germ-free mice are transfaunated with preterm infant microbiota. This state-of-the-art experimental model will allow specific investigation of the impact of clinically relevant microbiota on brain development that is not possible in human infants. The goal of this proposal is to discover intestinal microbiome patterns associated with school readiness, identify the key time points that represent windows of opportunity for microbiome optimization, and identify mechanisms by which the intestinal microbiome impacts brain development and behavior. This new knowledge will enhance our understanding of the gut-brain axis and lay the fo...