Fig. 4: Visualisation of membrane permeabilisation in Gram-negative bacteria with fluorescent vancomycin probe 9.
SR-SIM fluorescence imaging of vancomycin probe 9 in E. coli strains ATCC 25922 (a), K12 (MB4827) (b), and CFT073 (e), and mutant E. coli (DC2 (c), lpxC (d) and waaL (f)). Bacteria were stained with probe 9 (32 µg/mL, Green) and FM4-64FX (5 µg/mL, red, bacterial membrane). Scale bar = 2 µm. g, h Visualisation of membrane permeabilisation at different temperatures. Airyscan confocal super-resolution imaging of probe 9 in E. coli strain BW25113. Bacteria were stained with probe 9 (32 µg/mL, Green) and FM4-64FX (5 µg/mL, red, bacterial membrane) at 15 °C or 37 °C. Arrows indicate cells labelled with probe 9 at dividing septum and peptidoglycan at 15 °C. Scale bar = 5 µm. i, j Determination of membrane permeabilisation in E. coli (ATCC 25922) using vancomycin probe 9. Bacteria were treated with compounds for 1 h at 37 °C, followed by labelling with the probe Vanco-NBD 9 (32 µg/mL), then left for 30 min at 37 °C. The samples were then measured by flow cytometer or Tecan plate reader. i Flow cytometry method. j Tecan plate reader method. The data are presented as the mean ± SEM (n ≥ 2). k–o Microfluidic single-cell analysis of probe accumulation in E. coli. Accumulation of vancomycin probe 9 in E. coli in M9 medium drug milieu at an extracellular concentration of 46 µg/mL in k the absence and l the presence of polymyxin B at an extracellular concentration of 1 µg/mL delivered to n = 241 and n = 232 individual E. coli, respectively. In both figures, data were collated from biological triplicate and fluorescence values were background subtracted and normalised by cell size. The symbols and shaded areas represent the mean and standard deviation of the corresponding single-cell values. The vertical dotted lines represent the time points at which the median of each dataset became larger than zero. The median remained zero throughout the entire experiments carried out with vancomycin-NBD 9 alone, hence the dotted line has been arbitrarily set at 11,100 s in k for comparison purposes only. Distributions of single-cell values for the kinetic parameters (m) t0, (n) k1 and (o) Fmax describing the accumulation of 9. The red dashed and blue dotted lines within each violin plot represent the median and quartiles of each data set, respectively. ****p value <0.0001. The fitting algorithm returned convergent transitions for the accumulation data of n = 46 (out of the 241 datasets available) bacteria treated with vancomycin-NBD 9 and for n = 161 (out of the 232 datasets available) bacteria cotreated with vancomycin-NBD 9 and polymyxin B. t0, k1 and Fmax values for single-cell accumulation profiles for which the fitting algorithm returned divergent transitions were arbitrarily set to 10,100 s, 0.01 a.u. s-2 and 0 for comparison purposes only. These values represent the time at which the experiments with both vancomycin-NBD and the combination of probe 9 and polymyxin B were terminated and the minimum values of k1 and Fmax measured across the two datasets, respectively.
