e. 10
mg/L). Cells were incubated with the antibiotic at 37°C for an additional 24 h, and then diluted 1:500 in LB to rid the culture of the antibiotic effect. The growth kinetics of both normalizers and treated cells were recorded using an automated selleck kinase inhibitor 96-well plate reader (Sunrise Tecan, Switzerland) at 37°C with 10 s of circular shaking every 15 min, followed by 10 s of settling at which time OD600nm was detected. The SGT for each sample was determined as the time when the OD600nm of the sample reached a threshold of 0.15 – 0.2. The relative size of the antibiotic tolerant persister subpopulation for each mutant’s culture was calculated as the log2 fold of change (-∆∆SGT) between the samples normalized to that of PA14. ∆∆SGT calculation We applied the methodology to calculate the ∆∆ct for quantitative polymerase chain reaction experiments (qPCR) [10, 11] by determining ∆∆SGT values
of samples compared to a calibrator. First, a ∆SGT value Ku-0059436 manufacturer was calculated for each sample according to the following equation: ΔSGT = (SGT Treated − SGT Normalizer) where the SGT of untreated normalizer cells was subtracted from the SGT of treated cells. Second, a ∆∆SGT value was calculated by subtracting the ∆SGT of the reference strain or condition (“calibrator”) from that of the sample: ΔΔSGT = (ΔSGT Sample − ΔSGT Calibrator). Fold change between the sample and the calibrator was calculated as: F = 2−ΔΔSGT . Results are presented as log2 fold changes: -∆∆SGT. Results and discussion Assessment of live bacteria cell number in a high throughput setting The SGT method is based on the time that a growing bacterial cell culture
takes to reach spectrophotometrically detectable levels being proportional to the starting bacterial inoculum [8]. This approach allows live bacteria within a culture to be quantified (Figure 1). The SGT of each sample is defined as the time required by the culture Smoothened to reach an OD600nm threshold that is set slightly above the detectable background at the start of the logarithmic phase of growth, 0.15-0.2 in the present study. Figure 1 SGT values are proportional to the initial inoculum. The linearity of SGT method was assessed in various strains and conditions. (A) Growth curves of the wild-type P. aeruginosa strain PA14 (PA) grown in LB (Green), LB + 3% Ethanol (Yellow) and in the defined medium M63 (Pink); PA14 isogenic mutant derivative cyt b1 (light blue); and wild-type strains A. baumanii (black) and E. coli DH5α (dark blue). (B) The time when the growth curves crossed the threshold (OD600nm = 0.15 – 0.2) is defined as the SGT. P. aeruginosa PA14 cells were grown to OD600nm = 2.0, when the concentration of cells was 4.07 x 109 ± 7.02 x 108 cells/mL according to CFU counts. The cells were diluted serially 1:10 in a 96-well plate reader to ODs below the detection threshold of the spectrophotometer, after which their growth kinetics was recorded and also determined at 18 h by CFU counts.