Fig. 1: Aminoglycoside and fluoroquinolone antibiotics induce cytoplasmic condensation in E. coli.

a Cytoplasmic condensation and/or lysis (yellow and red markers, respectively) induced by kanamycin (KAN), ciprofloxacin (CIP), and ampicillin (AMP) after 1–6 h of treatment (KAN: 3 h, CIP: 6 h, AMP: 1 h) in a population of wild-type, log-phase Escherichia coli cells, imaged in phase contrast. Times were chosen to reflect timescales of phenotypic change. Here and below, all antibiotic concentrations used were 10× MIC, while control cells were untreated; working MICs were 5 μg/mL for kanamycin, 0.1 μg/mL for ciprofloxacin, and 10 μg/mL for ampicillin. Ampicillin-treated cells exhibited membrane bulging and lysis, in contrast to cytoplasmic condensation and lysis observed in kanamycin- and ciprofloxacin-treated cells (Supplementary Fig. 1). Ciprofloxacin-treated cells also displayed filamentation. Cytoplasmic condensation and lysis were general across cells in a population, antibiotic concentrations ranging at least from 1× to 100× MIC, different aminoglycoside and fluoroquinolone antibiotics, and in Bacillus subtilis (Supplementary Figs. 3 and 4). Results are representative of three biological replicates in each condition. Scale bar, 10 μm. b E. coli cells with fluorescent genetic outer membrane (green fluorescent protein, GFP), inner membrane (mCherry), periplasmic (GFP), and cytoplasmic (mCherry) markers under ciprofloxacin treatment, imaged in phase contrast and epifluorescence. For similar cells treated with kanamycin, see Supplementary Fig. 3. Results are representative of three biological replicates for each marker, and yellow markers highlight condensed cells. Scale bars here and below, 3 μm. c Schematic of cytoplasmic condensation and lysis in E. coli treated with kanamycin and ciprofloxacin. d Fractions of all cells that are condensed or lysed. Error bars indicate one standard deviation, and data are presented as mean ± SEM. Data are derived from two different fields of view with at least 100 cells each (kanamycin, 133 and 149 cells; ciprofloxacin, 255 and 140 cells) and representative of two biological replicates. Note that cells may condense and lyse within the same hour; such cells are reflected only in lysed cell counts. The total condensed fraction counts all cells that are, or have previously been, condensed. e Hyperosmotic shock measurements in control and antibiotic-treated E. coli, of which three characteristic phase-contrast microscopy images are shown (top); here, ciprofloxacin-treated cells are shown. The percentage length contraction, an indicator of cellular turgor, is the percentage change in cell length immediately after hyperosmotic shock. Red dashed lines represent visual guides. The number of cells in each group are indicated in parentheses. Here and below, a box plot indicating the median (center), 25th and 75th percentile (bounds of box), and extreme data points not considered outliers (bounds of whiskers) is shown, and red crosses indicate outliers including the minimum and maximum values. p-values for two-sample Kolmogorov-Smirnov tests are shown next to corresponding brackets. f Atomic force microscopy (AFM) of cells. (Left) Phase-contrast and AFM topography image of a condensed, ciprofloxacin-treated cell. (Right) Inferred elastic moduli corresponding to the cellular regions shown to the left, as indicated by different colors, as well as along the cellular body of an untreated control cell. The number of cells in each group are indicated in parentheses. Box plot features and statistical tests refer to those of panel (e). For measurements for kanamycin-treated cells, see Supplementary Fig. 7. g, h Fluorescence intensities of control and antibiotic-treated E. coli in the presence of SYTOX Blue (g) and DiBAC₄(3) (h). The number of cells in each group are indicated in parentheses. Box plot features and statistical tests refer to those of panel (e). i Representative timelapses of ciprofloxacin-treated E. coli stained by SYTOX Blue and DiBAC₄(3), imaged in phase contrast and epifluorescence. Times show duration after the first image, and results are representative of three biological replicates.