Supplementary Figure 7: Phenotypic analysis of ciprofloxacin-sensitive gene deletion mutants. | Nature Methods

Supplementary Figure 7: Phenotypic analysis of ciprofloxacin-sensitive gene deletion mutants.

From: A multiplexable assay for screening antibiotic lethality against drug-tolerant bacteria

Supplementary Figure 7

a, Example TUNEL staining as observed by flow cytometry of wild-type E. coli treated with hydrogen peroxide during exponential phase (left), ciprofloxacin during exponential phase (middle), or ciprofloxacin during stationary phase (right). This is a representative experiment of three biologically independent experiments. In all panels, gray regions show untreated control samples, and blue regions show treated samples. Note the difference in TUNEL stained populations of E. coli with ciprofloxacin between exponential phase and stationary phase. b, DNA double-strand breaks induced by ciprofloxacin during exponential growth. Cells were grown to exponential phase in LB and treated with 100 ng/ml ciprofloxacin (–C) or vehicle for 1 h prior to TUNEL analysis by flow cytometry (mean ± s.d.; n = 3 biologically independent experiments, except for YibP, where n = 2 biologically independent experiments). Hydrogen peroxide (100 mM for 10 min) was used as a positive control. ch, Micrographs of E. coli strains treated with ciprofloxacin during exponential phase and stationary phase. These are representative images of two biologically independent experiments. For exponential phase images, cells were grown to OD ~0.1 and treated with ciprofloxacin (100 ng/ml) for 24 h. For stationary phase images, cells were grown to saturation for 24 h and treated with ciprofloxacin (1 µg/ml) for 24 h. Cells were then fixed, stained with FM4–64, and imaged. Note the lack of filamentation by ciprofloxacin against resource-depleted stationary phase cells, despite their sensitivity to killing. i, 24-point MIC curves of wild-type E. coli and E. coli harboring a gyrA S83A ciprofloxacin-resistance mutation. Overnight cultures were inoculated 1/10,000 into fresh LB media in final volumes of 100 µl in 96-well microtiter plates, and ciprofloxacin was added to the concentrations indicated. Cultures were incubated until untreated control cultures reached stationary phase, and optical density at 600 nm was measured (mean ± range; n = 2 biologically independent experiments). j, 24-point MIC curves of ΔyfgL E. coli and ΔyfgL E. coli harboring a gyrA D87Y ciprofloxacin-resistance mutation. Overnight cultures were inoculated 1/10,000 into fresh LB media in final volumes of 100 µl in 96-well microtiter plates, and ciprofloxacin was added to the concentrations indicated. Cultures were incubated until untreated control cultures reached stationary phase, and optical density at 600 nm was measured (mean ± range; n = 2 biologically independent experiments). Note that gyrA S83A and gyrA D87Y confer the same level of resistance. k, Sensitivity of the four strains shown in i and j to killing by ciprofloxacin in exponential phase. Cells were grown to ~107 CFU/ml and treated with ciprofloxacin for 24 h prior to plating (mean ± s.d.; n = 3 biologically independent experiments). l, Growth kinetics of the four strains in i and j. Overnight cultures were inoculated 1/10,000 into fresh LB media in final volumes of 100 µl in 96-well microtiter plates. Optical density at 600 nm was read every 10 min over 24 h, and curves were plotted (mean ± s.d.; n = 3 biologically independent experiments). m, 12-point kill curves of the four strains in i and j. Cultures were grown to stationary phase in LB media in final volumes of 100 µl in 96-well microtiter plates. Ampicillin was introduced at the concentrations noted, and cells were incubated for an additional 24 h prior to plating (mean ± range; n = 2 biologically independent experiments). Wild-type E. coli is shown in blue, and ΔyfgL E. coli is shown in red. Circles represent wild-type gyrA strains, and squares represent ciprofloxacin-resistant gyrA strains. Ampicillin requires high metabolic activity for killing, and thus these data support the notion that all strains were in stationary phase prior to the introduction of ciprofloxacin.

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