Fig. 3: Inferred XR and CS interactions are validated with high accuracy by experimental evolution.

a, Schematic of benchmarking conducted for 70 drug pairs by experimental evolution and IC₉₀ measurements. Twelve lineages were evolved in parallel for five passages in increasing concentrations of 23 antibiotics. At each passage, the culture growing at the highest concentration was transferred to a new antibiotic gradient. The IC₉₀ of the final resistant population was then measured for all lineages in the relevant antibiotics. b–d, Heatmaps of 70 new and known drug-pair interactions, split depending on whether they were inferred as CS (b), neutral (c) or XR (d). Interactions were tested in both directions, with the drug for which selection occurred shown first and the drug for which MIC/IC₉₀ was tested shown second. In each interaction, all tested lineages are shown (n = 9–12). Coloured boxes denote the interaction observed for a given lineage. The three columns on the right of the lineage results represent the summary for all lineages. We considered an interaction as validated if the log₂-transformed IC₉₀ fold change was >1 for XR and <−1 for CS in any direction tested for at least one lineage compared with the wild type. An interaction of a drug pair was deemed to be XR if there was at least one lineage showing XR despite any CS for other lineages. Interaction monochromaticity (that is, whether the interaction is exclusively CS or XR; neutral lineages do not affect this call and were labelled as not applicable (N/A)) and directionality (drug pair interacting consistently in both directions) are shown. Interactions referred to as reclassified in the text are those for which our inference and validation agree but previous reports have reported differently. The interaction in red (least monochromatic interaction) is used in Fig. 5 to understand the mechanisms in play. The interactions in bold are used later in Fig. 6 to test resistance evolution in drug combinations. The interaction in italics (drug pair 14), which was conflicting across studies (XR in one study and CS in another), has been inferred and validated to be CS.

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