MIC Determinations

Master plates of each bacterial strain were prepared in a 96-well plate format using overnight cultures in ~15% glycerol (~5 x 10⁸ CFU/mL) and stored at −80°C. The master plates were thawed prior to experiments and then used to inoculate the antibiotic plates with a pinner (VP Scientific, VP409), which transfers ~5 x 10⁴ CFUs into each well containing ~200 μL of growth media. MIC values were determined using either end-point (final OD600) analysis or calculating the AUC [47]. Briefly, for end-point MIC determination (Figs ​(Figs11 and ​and33 and S2 and S3 Figs), the 96-well plates were incubated for 22 h in a shaker operated at 37°C and then OD600 of each plate was measured using a plate reader (Infinite M200 Pro, Tecan). For each strain and antibiotic pair, the MIC value was defined as the lowest antibiotic concentration at which the final OD600 was below ~0.04 after background correction, which is slightly above the lower detection limit of our plate reader. Preliminary experiments were conducted with four replicates for clindamycin and fusidic acid. The remaining antibiotics were run twice with biological replicates. A Pearson correlation coefficient test was used to confirm the repeatability of the measurements and the p-values for MIC reduction significance were calculated using Wilcoxon rank sum test. For MIC determination using AUC values, plates were incubated under similar environmental conditions, but in an automated robotic system so that OD600 of growing cultures were recorded as a function of time (Fig 2D and 2E). Linear interpolations of the resulting growth curves (OD600 versus time, Fig 2D) were then integrated to calculate the AUC as a metric for growth using a custom MATLAB code (r2016a, MathWorks). MIC values were defined as the concentration of antibiotic where the AUC was reduced by at least 95% compared to the AUC without antibiotic. Although both methods gave qualitatively similar results, we used the AUC method whenever possible because it is more robust to experimental noise [47].