Extended Data Figure 6. Assessing potential confounding factors for the killing capacities of erythromycin, azithromycin and doxycycline.

a. Scatter plot of individual bacterial growth rates (μ) and percentage survival after a 5-hour treatment with 5-fold MIC of erythromycin, azithromycin or doxycycline. ris the Spearman correlation coefficient. Tested species are color-coded here and, in all panels thereafter as indicated at the bottom of this figure.

b. B. fragilis (blue), F. nucleatum (beige), P. copri (pink) and E. coli ED1a (grey) survival was assessed after a 5h erythromycin and azithromycin treatment (5-fold MIC) at 30°C (slow growth) and 37°C (fast growth) - mean±SD of three independent experiments. No monotonic trend was observed.

c. Scatter plot of MICs and % survival after a 5h treatment with 5-fold MIC of erythromycin, azithromycin or doxycycline. r is the Spearman correlation coefficient. Doxycycline exhibited a significant (p-value=0.0015) anti-correlation, i.e. more sensitive species to doxycycline (lower MIC) survived better when treated with antibiotic. Therefore, we tested further whether increasing the drug concentration in sensitive strains increased killing (panel d).

d. B. fragilis (blue) and F. nucleatum (beige) survival after a 5-hour treatment as function of increasing doxycycline concentrations (mean±SD of three independent experiments). No significant differences observed. In all cases doxycycline remained bacteriostatic. Significance calculated by unpaired two-sided t-test here and in all panels thereafter.

e. To evaluate whether outgrowth from stationary phase affected our results, we selected two slow-growing strains, E. rectale (green) and R. intestinalis (orange) and grew them for 2 or 3h after diluting from an overnight culture to an of OD₅₇₈ 0.01. Both strains were then treated for 5h with 5-fold MIC of erythromycin, azithromycin or doxycycline and their survival was assessed (mean±SD of three independent experiments). Although 3h grown cultures were killed slightly more effectively (difference is not statistically significant due to low number of replicates), this did not change the bactericidal or bacteriostatic characteristic of antibiotics. If anything, this means that we underestimate the killing for slow-growers, since all other experiments were performed with 2 h outgrowth. Nd: not detected (detection limit: 1 CFU/ml.)

f-g. The survival of 8 selected gut microbes was measured after treating cells in exponential phase (E – 2h after dilution from an overnight culture) or in stationary phase (S – overnight growth) with 5-fold MIC of erythromycin (f) or doxycycline (g) for 5h (mean±SD of three independent experiments). Consistent with the knowledge that antibiotic killing requires active growth, survival is higher in stationary phase for most strains (but not all – see F. nucleatum) that erythromycin or doxycycline kills. ns = non-significant; *, ** and *** denote p-value <0.05, <0.01 and <0.001, respectively. nd as in e.

h. E. coli ED1a survival was assessed after 5h treatment with 5-fold MIC of doxycycline in the presence or absence of oxygen. Killing was similar in both conditions.