Extended Data Figure 7. Identification and validation of macrolide and tetracycline antagonists (antidotes) in B. vulgatus and B. uniformis.

a. Schematic illustration of combinatorial screen concept: searching for antidote compounds that antagonize the antibacterial effect of erythromycin or doxycycline on commensal but not on pathogenic bacteria.

b. Z-scores on bacterial growth for combinatorial drug exposure with antibiotic and 1197 FDA-approved drugs of Prestwick library (2 replicates). Compounds that successfully protected B. vulgatus and/or B. uniformis in the presence of antibiotic (z-score > 3) are indicated in gray. The strongest hits (circles) were validated in concentration-dependent assays (c-d). Box plots as in Fig. 1c.

c. Validation of the strongest antagonistic interactions in independent experiments. Erythromycin and doxycycline concentrations were kept constant for each species and concentration ranges were tested for antagonist. Asterisks indicate that at least 25% of the bacterial growth (compared to no drug controls) could be rescued by the antagonist at a given concentration. Heat map depicts median growth across triplicates.

d. For 10 of the validated antagonists, 8 x 8 checkerboard assays were performed to define better the range of the antagonistic interaction. Heat maps depict bacterial growth based on normalized median of AUCs of 3-4 replicates. Antagonistic interactions are framed in red (all).

e. Percentage of surviving B. vulgatus cells were determined after 5h incubation with either erythromycin (3.25 μM) or doxycycline (0.4 μM) alone or in presence of benzbromarone (40 μM), dicumarol (20 μM), tolfenamic acid (40 μM) or diflunisal (80 μM). Data is based on three independent experiments. Boxplots are plotted as in Fig. 1c.