The spread of antibiotic resistance is attracting increased attention to combination-based treatments. Although drug combinations have been studied extensively for their effects on bacterial growth1–11, much less is known about their effects on bacterial long-term clearance, especially at cidal, clinically relevant concentrations12–14. Here, using en masse microplating and automated image analysis, we systematically quantify Staphylococcus aureus survival during prolonged exposure to pairwise and higher-order cidal drug combinations. By quantifying growth inhibition, early killing and longer-term population clearance by all pairs of 14 antibiotics, we find that clearance interactions are qualitatively different, often showing reciprocal suppression whereby the efficacy of the drug mixture is weaker than any of the individual drugs alone. Furthermore, in contrast to growth inhibition6–10 and early killing, clearance efficacy decreases rather than increases as more drugs are added. However, specific drugs targeting non-growing persisters15–17 circumvent these suppressive effects. Competition experiments show that reciprocal suppressive drug combinations select against resistance to any of the individual drugs, even counteracting methicillin-resistant Staphylococcus aureus both in vitro and in a Galleria mellonella larva model. As a consequence, adding a β-lactamase inhibitor that is commonly used to potentiate treatment against β-lactam-resistant strains can reduce rather than increase treatment efficacy. Together, these results underscore the importance of systematic mapping the long-term clearance efficacy of drug combinations for designing more-effective, resistance-proof multidrug regimes.
Bibliographical noteFunding Information:
We thank R. Gross, E. Shaer-Tamar, I. Yelin, M. Datta, D. Ross and M. Lukacisinova for reading the manuscript and for comments and suggestions; the members of S. Walker’s laboratory for bacterial strains; A. Horswill for the fluorescence plasmids; I. Yelin for the β-lactamase plasmid; and D. Ment for the G. mellonella larvae. V.L. was supported by a postdoctoral fellowship from the Human Frontier Science Program Organization (LT001011/2017-L) and in part by Technion Postdoctoral Fellowship. This work was supported in part by the US National Institutes of Health grant R01-GM081617, the ISRAEL SCIENCE FOUNDATION (grant no. 455/19) and the European Research Council FP7 ERC grant 281891 (to R.K.).
© 2022, The Author(s), under exclusive licence to Springer Nature Limited.