Extended Data Fig. 1: Analysis of Salmonella in vitro.
From: Limited impact of Salmonella stress and persisters on antibiotic clearance
(a) Sorting of Salmonella cells with different sizes. Salmonella expressing gfp under control of the chromosomal PsifB promoter61 showed correlated sideward-scatter (a proxy for size73) and green fluorescence signals. The combination of the two detection channels increased resolution and enabled enrichment of subsets by flow cytometric sorting using the gate boundaries shown as dashed red lines. (b) Growth curves in lysogeny broth in 96-well plates. Means and SDs for 4 (wild-type, WT), 5 (hipAD88N), or 6 (shpBQ97*) biological replicates (each measured in technical triplicates) are shown (OD600, optical density at 600 nm; Det. Lim., detection limit). (c) Division rate distributions of Salmonella strains in different mouse genotypes (data for wild-type Salmonella in SLC11A1r mice from4, n = 61,137 cells from seven independently infected mice; SLC11A1s, WT n = 34,851 from three independently infected mice; SLC11A1s, hipAD88N n = 832 from three independently infected mice). The inset shows medians for the individual mice (SLC11A1s WT/hipAD88N n = 3; SLC11A1r, WT n = 7; ANOVA with comparison against SLC11A1s, WT; P-values adjusted for multiple comparisons according to Holm-Šídák). (d) Survival of Salmonella strains in mouse spleen 1 h after administration of 0.1 mg enrofloxacin. Each circle represents an individual mouse (SLC11A1s: WT n = 10; hipAD88N n = 4; SLC11A1r, WT n = 7) two-tailed t-test, P-values adjusted for multiple comparisons according to Holm-Šídák). (e) Analysis of Salmonella/pRecA-mCherry fluorescence images. Detection of RecA-mCherry foci was enhanced by 2D Laplacian of Gaussian filtering (approximating a second derivative) of red fluorescence and false-color visualization. (f) Fraction of cells with no long-lasting RecA focus (“undamaged fraction”) during growth without enrofloxacin (Control, 146 cells), or exposure from t = 0 h to 1.5 or 5 mg/L enrofloxacin (130, 250 cells). The dotted lines represent monoexponential fits. The data for 5 mg/L are also shown in Fig. 3c. Summary data for the 5 mg/L exposure and an independent replicate are shown in Fig. 3d. (g) Flow-cytometry analysis of sideward scatter as a read-out for cell size73 of Salmonella before and after 1 h exposure to enrofloxacin during exponential growth in lysogeny broth (LB), slow growth in tissue-mimicking chemostats, or in mouse spleen. Each symbol represents an independent culture or an individual mouse (LB +/− n = 3; Chemostat +/− n = 5; Mouse spleen +/− n = 3; two-tailed t-test of log-transformed data with Holm-Šídák correction for multiple comparisons). (h) Snap-shots of Salmonella growing in nutrient-poor minimal medium (MM) without enrofloxacin and then in lysogeny broth (LB; video S1). (i) Fraction of undamaged cells before and after switching to LB, and dividing Salmonella after the LB switch (146 cells; LoD, limit of detection). RecA foci during the first 4 h are also shown in panel f (‘Control’). The dotted line is a monoexponential fit for damage before the LB switch. (j) Time difference between detection of a DNA double-strand bread (DSB) based on a long-lasting RecA focus, and re-initiation of cell division. Control cells without enrofloxacin exposure that were switched to lysogeny broth (No ENR/LB: DSB- n = 40; DSB+ n = 69), cells exposed for 1 h to enrofloxacin followed by a switch to LB (1 h/LB n = 35), and cells exposed to declining concentrations of enrofloxacin over 7 h followed by continuing incubation in nutrient-poor medium (‘in vivo-mimicking’, IVM n = 115) are shown. Each circle represents an individual cell. The statistical difference between groups was test using the Kruskal-Wallis-test with Dunn’s correction for multiple testing.
