Fig. 3: Gradual shifts in gut microbiome composition in response to ETI are driven by antibiotics and steroids, including Escherichia decline linked to reduced intestinal inflammation.

a Stool microbiome composition shifts from baseline (PERMANOVA on BC-dissimilarities, stratified by donor, adjusted for sex and age: Months from ETI start R2 = 2.3%, p = 0.001). PCoA shows first two dimensions; arrows connect each sample to its baseline. Colors indicate months from ETI treatment start: black=baseline (N = 34), yellow=3 months (N = 26), blue=6-12 months (N = 76), red=15-18 months (N = 40), green=21-24 months (N = 31). Geometric markers show mean shift from baseline. b Microbiome variance (R2) explained between sampling time points (PERMANOVA on BC-dissimilarities, stratified by donor). Circle size reflects R2; color indicates p-value, details in Supplementary Data 17. c Relative abundance of Escherichia-Shigella in stool samples across time and controls. Significance vs. baseline assessed by LME (ID as random factor). FDR < 0.1 (.), FDR < 0.05 (*), FDR < 0.01 (**), and FDR < 0.001 (***), values in Supplementary Data 7. Box plots show the median (line), IQR (box), 1.5× IQR range (whiskers), and outliers (points beyond whiskers). Sample time points are color-coded. d Microbiome variance (R2) explained by individual covariates. Blue: significant in simple, purple: in multiple PERMANOVA on BC-dissimilarities (p < 0.05), details in Supplementary Data 18. e Alpha diversity metrics (calculated on rarefied reads to minimum sequencing depth) significantly associated with clinical metadata. Heatmap hues represent signed effect sizes (Spearman’s rho or Cliff’s delta). FDR < 0.1 (.), FDR < 0.05 (*), FDR < 0.01 (**), and FDR < 0.001 (***). Features shown have effect size > |0.2 | . Details in Supplementary Data 8. f Fecal calprotectin negatively correlates with Escherichia-Shigella abundance. Sample time points are color-coded. Effect size= 0.31, FDR = 0.04, without confounders reported by metadeconfoundR. line =linear fit; shaded areas = 95% confidence intervals. g CFTR modulators inhibit growth of several gut microbes in vitro. Antibiotics were tested as controls. IC₂₅ = concentration for 25% growth inhibition after 22 h drug exposure compared to growth in 1% DMSO (i.e., untreated control). “<“ = IC25 below tested; “>“ = IC25 above tested dose.