Measurement of persistence

Cells were grown in 20 ml of M9 medium with glycerol containing chloramphenicol (25 μg/ml) and ampicillin (25 μg/ml) to the exponential phase. At OD_600nm of around 0.4, hipA was induced with 0.2% arabinose for 95 min. Cultures were then treated with ciprofloxacin (2 μg/ml; Sigma-Aldrich) for 5 hours. For determination of colony-forming units (CFU), 1 ml of aliquots was taken before arabinose addition, 95 min after hipA induction, and 5 hours of ciprofloxacin treatment. Cells were washed with phosphate-buffered saline, serially diluted, plated on LB agar plates containing 0.4% glucose, and grown for 24 to 40 hours at 37°C. Persistence was calculated by dividing the number of CFU/ml of ciprofloxacin-treated culture with the CFU/ml of the culture before antibiotic addition and presented as a frequency of surviving (persister) cells in log₁₀ scale.