NICU site and infant age at sampling drive gut microbial and metabolite composition
OPACITY
After adjusting for covariates, infant DOL at sampling had the greatest impact on alpha diversity with significant increases in VOC and metabolite richness (p<0.03) as well as bacterial richness (p=0.03) and Shannon diversity (p<0.001) observed over time (Line chart illustrating alpha diversity of stool bacterial communities (blue), stool volatile organic compounds (VOCs) (green), and stool and urine metabolites (gold) on the y-axis, correlated with day of life (DOL) of infants on the x-axis. Lines on each panel represent mean alpha diversity observed in lactoferrin (dark) and placebo (light) cohorts. Shaded areas around each line represent 95% CIs. Numbers of samples included in each analysis are indicated in plot header (n).Lactoferrin exposure explained a mean of 3% of the variance between gut microbiota and metabolite sample compositions across all timepoints. NICU site had the greatest influence, describing a mean 30% variance across all timepoints (Bar chart demonstrating the impact of clinical variables (grouped on the y-axis) on stool bacterial (blue), stool volatile organic compound (VOC) (green), and stool and urine metabolite composition (gold). Impact is illustrated as explained variance (x-axis). Variables exerting greater influence on sample composition thus have larger bars. Significant associations (p<0.05) are highlighted with an asterisk. One sample per patient per timepoint was included in longitudinal analysis from earliest days of life (DOL) samples (darkest colour) to latest DOL samples (lightest colour). Numbers of samples included in each analysis are indicated in plot header (n). Further details of samples included per timepoint are available in samples per timepoint (figure), Despite the lack of overall impact of lactoferrin supplementation, analysis with MaAsLin2 identified some bacterial genera and metabolite features significantly associated with clinical features, including lactoferrin exposure. Models were built to control for clinical covariates including infant age at sampling, health status, feed type, birth weight, gestational age and days of exposure to trial intervention (lactoferrin or placebo). Due to the significant impact of NICU site on sample compositions (Relative abundances of several (19) bacterial and metabolite (1664 stool, 321 urine) features were significantly different between trial arms (Heatmap showing the impact of lactoferrin supplementation on relative abundance of bacterial genera (y-axis) across each individual Neonatal Intensive Care Unit site (x-axis). Colour is used to illustrate positive (green) and negative (red) associations with either lactoferrin receipt (left panel) or increasing intervention days (right panel). Null associations remain blank. NICU sites administering probiotics are highlighted purple on the x-axis.In combined analysis from every NICU site controlling for all clinical factors, proportional abundance of only three unidentified stool metabolites (p>0.005, Q>0.20) and acetic acid (p=0.02, Q=0.25) were significantly higher in infants receiving lactoferrin, while one single unidentified stool metabolite (p=0.006, Q=0.24) and 2-methyl-propanal (p=0.01, Q=0.21) were significantly higher in placebo. No bacterial genera remained significantly associated with either trial arm (Volcano plot illustrating combined bacterial, volatile organic compound (VOC) and metabolite features associated with lactoferrin supplementation across all Neonatal Intensive Care Unit sites. Each point represents a single feature, coloured by omics method. Higher effect sizes represent greater relative abundance of each feature in lactoferrin receipt cohort, lower effect sizes represent reduced relative abundance of each feature in lactoferrin receipt cohort. Features with significantly altered relative abundance identified by MaAsLin2 (following Benjamini-Hochberg correction) are highlighted with greater opacity and labelled with feature names where available.