With an OD600nm
threshold of 0.15, ∆SGT values were calculated as: ΔSGT = (SGT Treated (meropenem) − SGT Normalizer (untreated)) for each sample. The see more relative size of the antibiotic tolerant KU55933 persister subpopulation in each mutant’s culture was calculated as the log2 fold of change (−∆∆SGT) where: ΔΔSGT = (ΔSGT Sample (mvfRor pqsBC)) − ΔSGT Calibrator (PA14)). Figure 2 Example of SGT method use: assessment of the relative bactericidal activity of meropenem on various P. aeruginosa isogenic mutants. (A) Wild-type PA14 (blue) and its isogenic mutant derivatives mvfR (black) and pqsBC (red) were grown to mid-logarithmic phase before being subjected to a 24 h treatment with meropenem (10 mg/L) at 37°C (no meropenem added to normalizers). Following 1:500 dilution, the growth kinetics of normalizers and treated samples were recorded. Employing an OD600nm = 0.15, ∆SGT values were calculated as the difference between treated and normalizer SGTs. ∆∆SGT values were calculated as
the difference of between ∆SGTs of the mutants to that of wild-type PA14, which served as the calibrator. (B) For the SGT method, log2 fold of change was calculated as -∆∆SGT (empty bars). For CFU counting, normalizers and treated cells were serially diluted and plated. For comparison purposes, CFU count results are also presented as log2 fold of change (filled bars). The differences between the values obtained by the two methods did not differ significantly (p > 0.1). The mvfR mutant cells had a lower number (log2 fold change of −3.0 ± 0.29) and pqsBC mutant cells had a higher number (log2 fold change of Selleckchem Regorafenib 2.1 ± 0.07) of surviving cells than wild-type PA14 cells (Figure Resminostat 2B). There was a strong concordance between these SGT data and CFU data obtained in parallel (p > 0.1), providing validation of the SGT method (Figure 2B). Example 2: Screening for a compound’s effect on the size of an antibiotic tolerant subpopulation Another practical application of the SGT method is screening for compounds that affect the formation of antibiotic tolerant cells. To demonstrate this application, we
examined the effects of four compounds on the size of persister subpopulations in PA14 cultures exposed to a lethal dose of meropenem (10 mg/L). Specifically, the compounds used were: (i) the HAQ precursor anthranilic acid (AA) ; (ii) the AA analog 3-AA; and the two antibiotics (iii) gentamicin and (iv) ciprofloxacin (Figure 3A). Figure 3 Example of SGT method use: assessment of the relative efficacy of compounds on the size of the persister cell fraction using the SGT method. (A) PA14 cells were grown to the mid-logarithmic stage (arrow) in the absence or presence of AA (0.75 mM), 3-AA (0.75 mM), gentamicin (Gent, 1.5 mg/L) and ciprofloxacin (Cipro, 0.04 mg/L). Meropenem was applied as in Figure 2. (B) A comparison of survival fraction sizes obtained by SGT (empty bars) and CFU counting (filled bars) methods, presented as log2 fold change.