Supplementary Figure 3: Screens of the E. coli Keio collection for killing and growth inhibition by ciprofloxacin. | Nature Methods

Supplementary Figure 3: Screens of the E. coli Keio collection for killing and growth inhibition by ciprofloxacin.

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

Supplementary Figure 3

a, Image and example analysis of the plate shown in Fig. 2a, depicting the contrast between highly fluorescent colonies (dead cells) and those that have been masked by the reduction of TTC to TPF (live cells). b, Replicate plot of normalized fluorescence values of the Keio collection in the absence of ciprofloxacin. In biologically independent duplicate experiments, colonies were seeded onto nitrocellulose membranes in 384-density and grown for 24 h. Membranes were transferred to LB plates without ciprofloxacin and incubated for an additional 24 h. Membranes were then transferred to LB supplemented with 50 µg/ml TTC for 1 h prior to imaging. Fluorescence values were normalized according to row, column, and plate as described in the Methods section. c, Replicate plot of normalized fluorescence values of the Keio collection treated with 1 µg/ml ciprofloxacin. In biologically independent duplicate experiments, colonies were seeded onto nitrocellulose membranes in 384-density and grown for 24 h. Membranes were transferred to LB plates containing 1 µg/ml ciprofloxacin and incubated for an additional 24 h. Membranes were then transferred to LB supplemented with 50 µg/ml TTC for 1 h prior to imaging. Fluorescence values were normalized according to row, column, and plate as described in the Methods section. d, Normalized data of the screen of the Keio collection for colony fluorescence using the SPOCK assay. The yellow region shows hit strains based on a 3σ hit cutoff. With this criterion, 38 strains were identified as being sensitive to killing by ciprofloxacin under resource-depleted, tolerant conditions. e, Replicate plot of normalized optical density values of the Keio collection in the absence of ciprofloxacin. In biologically independent duplicate experiments, strains were grown in liquid LB in 384-density for 18 h and read at 600 nm. Data were normalized according to row, column, and plate as described in the Methods section. f, Replicate plot of normalized optical density values of the Keio collection in the presence of ciprofloxacin. In biologically independent duplicate experiments, strains were grown in liquid LB supplemented with 1/8 × MIC ciprofloxacin in 384-density for 18 h and read at 600 nm. Data were normalized according to row, column, and plate as described in the Methods section. g, Normalized data of the screen of the Keio collection for growth inhibition in liquid culture. The blue region shows strains that displayed enhanced growth inhibition in the presence of ciprofloxacin based on a 1.5σ hit cutoff. 27 strains were identified as being ciprofloxacin sensitive.

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