Beppler, C.,Tekin, E.,White, C.,Mao, Z. Y.,Miller, J. H.,Damoiseaux, R.,Savage, V. M.,Yeh, P. J.

Background: In drug-drug interactions, there are surprising cases in which the growth inhibition of bacteria by a single antibiotic decreases when a second antibiotic is added. These interactions are termed suppressive and have been argued to have the potential to limit the evolution of resistance. Nevertheless, little attention has been given to suppressive interactions because clinical studies typically search for increases in killing efficiency and because suppressive interactions are believed to be rare based on pairwise studies. Results: Here, we quantify the effects of single-, double-, and triple-drug combinations from a set of 14 antibiotics and 3 bacteria strains, totaling 364 unique three-drug combinations per bacteria strain. We find that increasing the number of drugs can increase the prevalence of suppressive interactions: 17% of three-drug combinations are suppressive compared to 5% of two-drug combinations in this study. Most cases of suppression we find (97%) are "hidden" cases for which the triple-drug bacterial growth is less than the single-drug treatments but exceeds that of a pairwise combination. Conclusions: We find a surprising number of suppressive interactions in higher-order drug combinations. Without examining lower-order (pairwise) bacterial growth, emergent suppressive effects would be missed, potentially affecting our understanding of evolution of resistance and treatment strategies for resistant pathogens. These findings suggest that careful examination of the full factorial of drug combinations is needed to uncover suppressive interactions in higher-order combinations.