Fig. 2: Analysis of L-Ara4N-modification levels of lipid A and their correlation with heteroresistance frequency and the number of cells that survived after colistin treatment.

a Heat map summarizing the MS-based analysis of L-Ara4N-modification levels of lipid A in selected E. xiangfangensis strains. Corresponding, representative mass spectra are shown in Supplementary Fig. 5, further details are specified in Supplementary Data 4. b General chemical structure of Enterobacter lipid A. c Heat map summarizing the MS-based analysis of L-Ara4N-modification levels of lipid A in 11 other Enterobacter species observed in this study. d Various Enterobacter isolates grown at identical conditions (LB broth, O.D.₆₀₀ 0.8–1.0, without colistin) were analyzed for L-Ara4N-modification of lipid A ((see panels a and c), Supplementary Data 4) and for their correlation with % of heteroresistant cells (on a plate containing 8 and 32 mg/L of colistin), and % of cells survived after colistin treatment (64 mg/L for 15 min). The % of heteroresistance CFUs at 8 and 32 mg/L was positively associated with L-Ara4N-modification levels of lipid A (Pearson’s correlation coefficient, r = 0.88 and 0.73, respectively). This data suggested colistin heteroresistance to be directly associated with such levels. Given that only a fraction of isogenic growing population survives on plates containing 8 and 32 mg/L of colistin, we determine the % of cells that can survive after colistin treatment (64 mg/L for 15 min) by Live/Dead^TM staining assay. This assay showed a positive association (r = 0.86) between % of cells survived and L-Ara4N-modified lipid A. This data indicated that only a certain number of cells from isogenic growing population carried modified lipid A and can survive when exposed to colistin, resulting in the heteroresistance phenomenon. This data show that cells with L-Ara4N-modified lipid A form the founder population of the colistin heteroresistance (also see Supplementary Table 5). Source data are provided as a Source data file.