Time-kill assays with Escherichia coli

Time-kill assays were conducted as previously described (Nordholt et al., 2021). E. coli were inoculated into 10 ml M9 to a defined number of cells (10⁴ cfu/ml) and incubated at 37°C with agitation at 220 rpm. After 24 h, OD₆₀₀ was determined and adjusted to an OD₆₀₀ of 0.01 (~10⁷ cfu/ml) in spent medium in 2 ml tubes (PP, LABSOLUTE) in a total volume of 900 μl. To maintain the cellular physiology in the stationary phase, time-kill assays were conducted in spent medium from the pre-culture, which was obtained by removing cells from the pre-culture medium by centrifugation for 2 min at 16000 g. After dilution of the cells, 10 μl were sampled to determine the initial cell concentration. Antimicrobials were added and cultures were incubated at 37°C with agitation (1,200 rpm) in a tabletop Thermomixer (STARLAB). Samples of 10 μl were taken and immediately serially diluted in 96-well microplates with 90 μl phosphate buffered saline (PBS, pH 7; 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, 2.68 mM KCl, 137 mM NaCl) per well. To enumerate colony forming units (cfu), 10 μl from each well were spotted on square LB agar plates. Plates were left to dry and incubated at 30°C for 16–24 h. To determine the fraction of survivors for single time point incubations, the initial time point before addition of antimicrobial and the final time point after 20 min of exposure were sampled.