Aminoglycoside Uptake Assay

Tobramycin (gentamicin, kanamycin or streptomycin) extraction coupled with cell growth inhibition was explored for antibiotic uptake assay as follows. Briefly, 1 ml persister cells, after hypoionic shock treatment in the presence of each antibiotic at concentrations as described in Supplementary Table S2, were washed twice with PBS and re-suspended in 100 μL cell wall-digestion buffer (30 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mg/mL lysozyme) for further incubation at room temperature for 2 h. Cells were subjected to three cycles of freezing treatment at −80°C, thermally denatured at 90°C for 10 min (Note: the bactericidal activity of each aminoglycoside after heating at 90^oC for 15 min was verified to be almost fully retained; refer to Supplementary Figures S5A, S6A) and centrifuged for removing cell debris and denatured proteins. Afterward, 5 μL supernatant was spotted on E. coli-seeded LB agar dish for further incubation at 37°C for 8–10 h and the diameter of cell growth inhibition zone was measured. In addition, tobramycin or gentamicin uptake by CCCP or FCCP pre-treated persister cells was measured similarly. A standard curve was prepared by directly adding each aminoglycoside at different concentrations (0, 15, 25, 50, 75, and 100 μg/mL) into persister cell suspension with the cell-wall digestion buffer. The tobramycin uptake by S. aureus cells was measured using the same procedure except of applying a different cell wall-digestion buffer (30 mM Tris–HCl, pH 8.0) plus 20 μg/mL lysostaphin [purchased from Sangon Biotech (Shanghai) Co., Ltd.; Cat no.: A609001].