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Updated: Jul 7, 2025

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations
Published on: July 24, 2021
The evolution of resistance to synergistic multi-drug combinations is more complex than evolving resistance to each
Natalie Ann Lozano-Huntelman1, Austin Bullivant1, Jonathan Chacon-Barahona1
1Department of Ecology and Evolutionary Biology University of California, Los Angeles Los Angeles California USA.
Abstract:
Multidrug antibiotic resistance is an urgent public health concern. Multiple strategies have been suggested to alleviate this problem, including the use of antibiotic combinations and cyclic therapies. We examine how adaptation to (1) combinations of drugs affects resistance to individual drugs, and to (2) individual drugs alters responses to drug combinations. To evaluate this, we evolved multiple strains of drug resistant Staphylococcus epidermidis in the lab. We show that evolving resistance to four highly synergistic combinations does not result in cross-resistance to all of their components. Likewise, prior resistance to one antibiotic in a combination does not guarantee survival when exposed to the combination. We also identify four 3-step and four 2-step treatments that inhibit bacterial growth and confer collateral sensitivity with each step, impeding the development of multidrug resistance. This study highlights the importance of considering higher-order drug combinations in sequential therapies and how antibiotic interactions can influence the evolutionary trajectory of bacterial populations.
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