Persister Assay

The frequency of S. aureus persister cells in growing cultures (type II persisters) was determined by bactericidal antibiotic killing of the susceptible population and enumeration of surviving cells. Cells from an overnight culture was diluted 1/1000 in 2 ml fresh TSB and incubated in 15 ml test tubes at 37°C and 200 rpm for 2.5 h to reach exponential growth and a cell density of approximately 1–4 × 10⁷ CFU/ml. An aliquot was taken to allow determination of the actual CFUs (by serial dilutions on TSA plates) and the culture was supplemented with a lethal concentration (20x MIC) of antibiotic. After 24 h of incubation, 1 ml of culture was withdrawn and centrifuged (12,000 g for 5 min), washed with 0.9% NaCl to remove the antibiotic, and plated on TSA. Plates were incubated at 37°C for 24 h and the persister frequency was calculated as the plate count relative to the CFUs obtained at time 0 h. The persister level was arbitrarily set to 0.5 CFU/ml if a sample had less than 1 CFU/ml, i.e., below the detection limit. Stationary phase persister cells (type I persisters) were evaluated by transfer of 24 h cultures to new test tubes followed by addition of 100x MIC of antibiotic and comparing the CFUs before and after another 24 h of incubation. For some assays stationary phase cells were supplemented with exogenous supernatants prior to antibiotic challenge. A minimum of three biological replicates (overnight cultures originating from individual colonies) were included for each assay and experimental condition. All experimental results are representative of repeated independent experiments. Representative colonies from the persister assay were isolated to verify the non-inherited nature of persistence by confirmation of their susceptibility toward the antibiotic with which they were isolated and an unaltered persister frequency in subsequent assays.