Extended Data Fig. 2: Salmonella analysis in vivo.
From: Limited impact of Salmonella stress and persisters on antibiotic clearance
(a) Traditional model and actual mechanism underlying rapid loss of colony-forming units. In the traditional models, a small subset of persisters, some of which are triggered by stresses, survive antibiotic exposure and give rise to colonies on plates. However, antibiotic exposure actually kills only few cells, but survivors are poisoned by drug, which remains bound to its target (depicted as yellow symbols). Most of these poisoned cells die after plating. Thus, colony counts reflect mostly post-exposure killing rather than viability loss during exposure. (b) Plasmid map of the SOS-reporter construct. The Pcad promoter is repressed by LexA36. LexA self-cleavage (SOS-response) is associated with unbinding of LexA to DNA, de-repressing Pcad. gfp-ova encodes a destabilized GFP variant to report current promoter activities. aphA encodes aminoglycoside phosphotransferase conferring resistance to kanamycin. repA encodes RepA which replicates plasmids with a oriSC101 origin (T, terminator). (c) Gating strategy for identifying Salmonella reporter cells in spleen homogenates based on the red fluorescence of chromosomally encoded mCherry (Ex, excitation wavelength; Em, emission range). mCherry-Salmonella were then analyzed for green fluorescence as shown in Fig. 3h. (d) Green fluorescence of Salmonella retrieved from mouse spleen 4 h after enrofloxacin administration and incubated for 3 h in lysogeny broth (LB). Control Salmonella without gfp are also shown. The data represent histograms pooled from 2 mice. (e) Colony recovery of GFP− (SOS−) and GFP+ (SOS+) Salmonella sorted from spleen homogenates from untreated mice (0 h), or from treated mice 1 h or 4 h after enrofloxacin administration. Survival was determined by plating on lysogeny-broth plates. Each circle represents an individual mouse (untreated n = 14; 1 h GFP +/− n = 6; 4 h GFP +/− n = 4). The statistical difference of GFP- and GFP+ contributions from treated mice was tested by two-way ANOVA (column factor, two-tailed, single comparison). (f) Survival of Salmonella strains in enrofloxacin-treated mice (hisGP69L: 1 h n = 10; 4 h n = 6; hisGP69L shpBQ97* 1 h/4 h n = 3; geometric means and geometric SDs; two-way ANOVA of log-transformed data). (g) Survival of Salmonella strains in lysogeny broth with enrofloxacin (geometric means and geometric SDs; six independent cultures from two different experiments; two-way ANOVA of log-transformed data).
