Experimental scheme of the larval zebrafish assays and sample collection. Conventional, mono-colonized, or mix5-colonized larval zebrafish were exposed at 5 dpf to water (control), DMSO (control) or 10 mg/L loperamide hydrochloride (treated) for 24 h and then transferred to water at 6 dpf. Samples were collected at 6 dpf, 7 dpf, and 11 dpf (T0, T1, T5) to measure fish growth and quantify bacterial community composition in all conditions (dpf, days post fertilization; CFUs, colony-forming units)

Loperamide affects conventional zebrafish microbiota as measured by 16S rRNA gene amplicons. A NMDS plot calculated using Bray–Curtis beta-diversity (k = 2) of percent normalized ASVs from 16S rRNA gene amplicons. Ellipse lines show the 95% confidence interval (standard deviation). Stress = 0.136; adonis2 PERMANOVA R² = 0.104; p = 0.029*. Only DMSO and loperamide-treated samples are shown. B Significant differentially abundant genera in loperamide-treated fish, compared to DMSO controls at each timepoint calculated using Limma (one against one) with conditions: relative log expression (RLE) normalized, effect log fold change > 2, Benjamini and Hochberg adjusted p-value < 0.05 (n = 5 per condition). Genera that occur at mean percent abundance > 1% are outlined in black and bold. C Beta-dispersion or within-condition dissimilarity index calculated using Bray–Curtis beta-diversity (n = 20). ****p < 0.001 for loperamide treatment, compared to DMSO. Wilcoxon test

In vitro survival in water of zebrafish-associated bacterial strains is affected by loperamide. Survival in water for 72 h after inoculation at 10⁶ CFUs/mL (mean ± standard deviation per condition is shown, n = 6–12: 2–4 independent assays of 3 biological replicates). *p < 0.05 for loperamide treatment, compared to DMSO. Wilcoxon test. Note log scale on y-axis

Loperamide can increase or reduce zebrafish mono-colonization. CFUs per fish of mono-colonized fish in A control conditions at T0 (6 dpf) ordered by colonization capacity and B after exposure to loperamide at 3 timepoints (n = 4 fish). Each point represents a single zebrafish (mean of 3 technical replicates). *p < 0.05 for loperamide treatment, compared to DMSO. Wilcoxon test. Note log scale for y-axis

Loperamide affects mix5-colonized gnotobiotic zebrafish bacterial load and composition. A Total CFUs per fish of mix5-colonized fish after exposure to loperamide at 3 timepoints (n = 3–4 fish). Each point represents a single zebrafish (mean of 3 technical replicates). No significant changes were found for loperamide treatment, compared to DMSO. Wilcoxon test. Note log scale for CFUs. B Percent abundance of each strain per mix5-colonized fish. Each bar is an individual fish sample. C Percent abundance of each strain in mix5-colonized fish per timepoint and treatment. Each point represents a single zebrafish (mean of 3 technical replicates). *p < 0.05 for loperamide treatment, compared to DMSO. Wilcoxon test

Simpson’s diversity of conventional and gnotobiotic zebrafish decreases but recovers after loperamide treatment. Alpha-diversity indices calculated at each timepoint for control water, DMSO, and loperamide-treated samples for A–C 16S rRNA gene amplicon data at the ASV level in conventional fish (n = 5 fish) and D–F CFUs per strain in mix5-colonized fish (n = 3–4 fish). A and D Simpson’s index (H). B and E Observed richness (S_obs) and C and F Pielou’s evenness (H/log(S_obs) are shown. Each point represents a single zebrafish with boxplots shown per condition. *p < 0.05 for loperamide treatment, compared to DMSO. Wilcoxon test