Fig. 5

Screening of resistance mutations associated with cross-resistance or collateral sensitivity. a Daptomycin selection (8 mg/L) of a pooled population of in vitro selected rifampicin-resistant clones. Survival was quantified by CFU counting on BHI agar plates at 3 h and 24 h of exposure. Error bars represent ± SEM of three independent exposures to daptomycin. b Rifampicin-resistant mutant quantification of rifampicin mutant before and after 3 h or 24 h of daptomycin exposure. Each bar of the histogram represents the averaged normalised count of the different rpoB mutants in the population. Average quantification of the three replicates at T = 0 and after daptomycin exposure are indicated by blue and red bars respectively. Bars are superimposed for each mutant and overlap of the bars are coloured in purple. Increases and decreases in allele frequencies after daptomycin exposure are indicated by red and blue bars respectively on the top of purple bars. c Volcano plot showing fold change in rpoB alleles frequency after 24 h of daptomycin exposure. Each dot represents a different rpoB mutant. Orange dots represent mutants with p value < 0.1 by Wald test. d Rifampicin resistance mutations associated with significant fold change after 24 h of daptomycin treatment. Mutations with positive and negative log2 fold change are predicted to be associated with cross-resistance and collateral sensitivity to daptomycin, respectively. The intensity of the blue coloration of the bars represents adjusted p values (Wald test). e Daptomycin time kill assays. Rifampicin-resistant mutants were assessed in triplicates (biological replicates), points represent the mean survival at each time point and error bars SD. Dashed lines represent detection limit