PROJECT SUMMARYSevere Pediatric Acute Respiratory Distress Syndrome (PARDS) is a life-threatening condition with highmortality (33%). Novel therapies to improve mortality in this condition are critical. Multiple retrospective studiesfrom our group and others have demonstrated an association between early enteral nutrition (EEN) anddecreased mortality in children with PARDS but mechanisms for this association are unclear. Crosstalkbetween the lung and gut microbiome is a potential mechanism by which EEN may reduce PARDS mortality.Diet can rapidly alter the relative abundance of beneficial butyrate-producing commensal gut bacteria toincrease fecal butyrate. In animal models of ARDS butyrate pre-treatment decreases lung inflammation andinjury. We hypothesize that in severe PARDS EEN increases relative abundance of butyrate producing gutcommensals thereby increasing butyrate levels and reducing acute lung inflammation and injury. EEN is anovel pathway to improve outcomes in these children. The PROSpect study a multi-center NIH-fundedclinical trial will randomize 1000 children with severe PARDS to compare positioning and ventilation strategiesto improve patient outcomes. This clinical trial presents a unique opportunity to investigate potentialmechanistic underpinnings of EEN as a targeted approach to improve outcomes for children with severePARDS. We will conduct our study as an ancillary study to the PROSpect study. The specific aims of ourstudy are: Aim 1:To test the hypothesis that relative abundance of butyrate producing fecal bacteriafecal butyrate and patient outcomes differ by EEN exposure in severe PARDS. We will obtain fecalspecimens from 180 patients on days 0-7 of mechanical ventilation to assess the effect of EEN and type ofEEN ( prebiotics) on the gut microbiome signature with 16S rRNA gene sequencing. We will assessdifferences in measured fecal butyrate and other short chain fatty acids (SCFA) by EEN and type of EEN. On asubset of fecal samples we will use whole genome shotgun metagenomics sequencing (WGS) to identifyspecies and strains of butyrate-producing commensal bacteria important in patients with PARDS. Aim 2: Totest the hypothesis that lower respiratory tract inflammation acute lung injury and innate immune cellgene expression patterns differ by fecal SCFA concentration in severe PARDS. We will obtainendotracheal aspirate specimens from patients in Aim 1 on PARDS days 0 and 3 to test associations betweenfecal SCFA and critical cytokines implicated in PARDS lung pathophysiology and key biomarkers of PARDSacute lung injury. We will utilize whole tracheal aspirate single nuclei RNASeq (snRNASeq) to compare geneexpression patterns for tracheal aspirate immune cell populations in patients by fecal butyrate. This study willimprove our understanding of the mechanistic underpinnings for how EEN may improve clinical outcomes ofPARDS. Loss of butyrate producing commensal bacteria may prove to be a critical diagnostic readout for riskassessment or to identify potential microbial-based treatment to improve outcomes for children with PARDS.