Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation
(A and B) We used time-lapse analysis of single cells to study tolerance to antibiotics. Bacteria were first grown in LB medium (marked blue), shifted to spent LB at 160 min (yellow), shifted to spent LB containing 0.05 µg/ml ciprofloxacin at 290 min (red), and shifted back to LB at 435 min (blue). (A) Bacterial cells were grown in microfluidic devices in dead-ended channels, with medium flowing through a bigger main channel orthogonal to them. The top row shows a temporal montage of images of a channel in which the bottom cell started expressing ttss-1 and resumed division after exposure to 0.05 µg/ml ciprofloxacin (“Ch 1”), and the bottom row shows a temporal montage of images of a channel in which the bottom cell did not express ttss-1 and did not resume division after exposure to antibiotic (“Ch 2”). For each channel, five sets of still images from different phases of the experiment are shown. Each set consists of 12 images recorded at 5-min time intervals. (B) Quantitative analysis of all 149 cells from this experiment. Every horizontal line represents data for an individual cell over 975 min. Dots mark the time points at which this cell divided. Cells are sorted according to ttss-1 expression during antibiotic exposure, measured as mean GFP fluorescence during that time interval. The cell index indicates the rank of a cell according to its mean GFP expression during antibiotic exposure; a lower cell index indicates lower GFP expression, and a higher cell index indicates higher GFP expression. The color of the lines indicates real-time GFP intensity. Shading indicates the data corresponding to the cells shown in (A). Three independent experiments were performed, all of them showing significant positive correlations of survival with ttss-1 expression (logistic regression with ANOVA, p = 8.8×10−14, 2.4×10−5, 1.3×10−8; N = 149, 137, and 144), indicating that T1+ cells preferentially survive antibiotic exposure. (C) Histogram of cells in different GFP categories. Color-coding of the columns denotes the probabilities to survive exposure to 0.05 µg/ml ciprofloxacin. The columns were assigned visually to two categories according to GFP intensity (“GFP on,” “GFP off”), and the percentage of cells surviving in the different categories was calculated. (D) Analysis of the single cell elongation rates 75 min before and 25 min after addition of antibiotic and survival. The black curve indicates the survival probability depending on the cell elongation rate as determined by a logistic regression model. The histograms show how many cells were in the respective ranges of elongation rates, and whether they survive antibiotic treatment (full bars, top) or die (empty bars, bottom). For all three experiments, survival was negatively correlated with single cell elongation rates (logistic regression with ANOVA, p = 7.8×10−4, 3.2×10−4, 1.0×10−4, N = 151, 137, and 114).