Fig. 2.
Cells isolated by cephalexin treatment and filtration are antibiotic-tolerant and regrow after treatment. a Time-kill curve of isolated cells treated with cephalexin (50 µg/ml) for 8 h in liquid medium (n = 5 biologically independent cultures). A uniphasic exponential curve was fitted onto the data with a killing rate (kp = 0.144) that is much lower than for susceptible cells (kn = 4.98; Supplementary Fig. 1a). The killing rate of persisters presumably reflects the rate of persister awakening in the presence of cephalexin (kp: killing rate of persisters). b Treatment of isolated cells with cephalexin (50 µg/ml) on an agarose pad supplemented with MHB shows that the majority of the cells is not affected by the antibiotic (scale bar: 10 µm). c Cells isolated by filtration start dividing on an agarose pad supplemented with MHB (scale bar: 10 µm). d Isolated cells display multidrug tolerance, a trait associated with persistence. Fraction of surviving cells after a 5-h treatment with cephalexin (50 µg/ml), aztreonam (0.64 µg/ml), cefsulodin (320 µg/ml), mecillinam (5 µg/ml), ampicillin (40 µg/ml), and ciprofloxacin (0.32 µg/ml), starting from an exponential phase culture or a sample consisting of isolated cephalexin persisters. For all tested antibiotics, isolated cells show a significantly higher tolerance as compared to exponential phase cells (cephalexin: p < 0.0001, n = 7 and n = 11 biologically independent cultures respectively; aztreonam: p = 0.004, n = 6 and n = 9; cefsulodin: p < 0.0001, n = 3 and n = 6; mecillinam: p < 0.0001, n = 5 and n = 9; ampicillin: p < 0.0001, n = 5 and n = 9; ciprofloxacin: p < 0.0001, n = 9 and n = 9). Whiskers represent 10–90 percentiles.