Figure 1: Evolution of antibiotic resistance under antibiotic cycling condition.

(a) Experimental design for bacterial evolution under cyclic exposure to two antibiotics. Wild-type E. coli was cultured in the morbidostat system with dynamic antibiotic concentrations. The Every t days (where t=1, 3 and 6), the drugs were switched in an alternating manner and the cell cultures were sampled daily and stored at −80 °C. After a total of 12 or 24 days the MICs of the collected samples were determined. (b) Evolutionary trajectories of KAN and POL resistance (green and yellow curves, respectively) under single antibiotic condition. Trajectories of parallel experiments were shown as pale lines and the average of parallel experiments was in solid line with empty circles. The resistance levels were measured as MIC fold change relative to the wild-type, calculated by log₂(MIC_i/MIC_WT), where MIC_i and MIC_WT are the MICs of the i-th day sample and wild-type E. coli, respectively. (c) Trajectories of KAN and POL resistance when cycled with 3 day interval. Pale lines indicate trajectories of parallel experiments and the average was shown as solid line with filled circles. The pale coloured background indicates the antibiotics used. (d,e) evolutionary trajectories of CHL and POL resistance (blue and yellow) under single antibiotic condition and cycling with 3 day interval, respectively. Similarly, for (f,g) CHL and NIT resistance (blue and purple), and (h) and (i) KAN and NAL resistance (green and orange). Samples sizes are n=3 for single antibiotic condition and n=2 for antibiotic cycling (biological duplicates).