a, Cartoon of possible modes of action for drug–drug interactions that function via modulation of the intracellular drug concentration. A given drug (antagonist, blue) inhibits the uptake or promotes the efflux of another drug (black), and thus decreases its intracellular concentration. b, Different antagonists (see Methods for concentrations) of gentamicin (red, 5 μg ml−1) and ciprofloxacin (yellow, 2.5 μg ml−1) identified in our screen for E. coli BW25113 also rescue the killing effect of the two bactericidal drugs in the same strain, or its parental MG1655 (top right and top left panels, respectively). With the exception of clindamycin (for gentamicin) and curcumin (for ciprofloxacin), all other antagonists decrease the intracellular concentration of their interacting drug (bottom panels). Gentamicin was detected by using radiolabelled compound, and ciprofloxacin with LC–MS/MS (Methods). The degree of rescue (top panels) in many cases follows the decrease in intracellular concentration (bottom panels), which implies that most of these interactions depend at least partially on modulating the intracellular concentration of the antagonized drug. c, Antagonisms are resolved in E. coli BW25113 mutants that lack key components that control the intracellular concentration of the antagonized drug. Aminoglycosides depend on proton motive force-energized uptake, and thus on respiratory complexes7,46; ciprofloxacin is effluxed by AcrAB–TolC29,47. For gentamicin, most interactions are resolved when respiration is defected, even the interaction with clindamycin (which does not modulate intracellular gentamicin concentration, see b); this presumably occurs because the mode of action and import of aminoglycosides are linked by a positive feedback loop7,48. For ciprofloxacin, antagonisms with paraquat and caffeine are resolved in the ΔacrA mutant, which implies that both compounds induce the AcrAB–TolC pump (well-established for paraquat49). By contrast, interactions with curcumin, benzalkonium and doxycycline remain largely intact in the ΔacrA mutant. The first interaction is expected, as curcumin does not modulate intracellular ciprofloxacin concentration (see b). In the other two cases, other component(s) besides AcrAB–TolC may be responsible for the altered ciprofloxacin import and/or export; for example, ciprofloxacin uses OmpF to enter the cell50. Ciprofloxacin and gentamicin concentrations were adjusted in all strains according to MIC (70% and 100% MIC for ciprofloxacin and gentamicin, respectively; all drug concentrations are listed in Supplementary Table 6). Bliss interaction scores (ε) were calculated as in the screen. Bar plots and error bars in b, c represent the average and s.d., respectively, across n independent biological replicates. d, Gentamicin and ciprofloxacin antagonism networks for E. coli BW. Nodes represent drugs coloured according to targeted cellular process (as in Extended Data Fig. 1a). Full and dashed edges represent antagonistic drug–drug interactions for which intracellular antibiotic concentration was and was not measured, respectively. Drug interactions that result in decreased intracellular concentration of the antagonized drug are represented by black edges. e, Quantification of antagonistic drug–drug interactions from the networks in (d). The bars for fluoroquinolones and aminoglycosides account for an extrapolation of antagonistic interactions to all other members of the two classes, assuming that they behave in the same way as ciprofloxacin and gentamicin, respectively. Source Data