Mapping the Role of AcrAB-TolC Efflux Pumps in the Evolution of Antibiotic Resistance Reveals Near-MIC Treatments Facilitate Resistance Acquisition

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Antibiotic resistance has become a major public health concern as bacteria evolve to evade drugs, leading to recurring infections and a decrease in antibiotic efficacy. Systematic efforts have revealed mechanisms involved in resistance; yet, in many cases, how these specific mechanisms accelerate or slow the evolution of resistance remains unclear. Here, we conducted a systematic study of the impact of the AcrAB-TolC efflux pump and its expression on the evolution of antibiotic resistance. We mapped how population growth rate and resistance change over time as a function of both the antibiotic concentration and the parent strain’s genetic background. We compared strains lacking functional AcrAB-TolC efflux pumps, the wild type strain, and those overexpressing the pumps. In all cases, resistance emerged when cultures were treated with chloramphenicol concentrations near the MIC of the parent strain. Strains grown in concentrations just above the MIC were the most prone to evolving high levels of drug resistance, in some cases reaching values that far exceed the concentrations they were treated with. The genetic background of the parent strain also influenced resistance acquisition. The strain overexpressing pumps evolved resistance more slowly and at lower levels than the wild type strain or the strain lacking functional pumps. In contrast, the wild type strain rapidly achieved resistance through mutations in pump genes and their associated regulators. Overall, our results suggest that treatment conditions just above the MIC pose the largest risk for the evolution of resistance, and precise control of pump expression levels accelerates this process.</jats:p><jats:sec><jats:title>IMPORTANCE</jats:title><jats:p>Combatting the rise of antibiotic resistance is a significant challenge. Efflux pumps are an important contributor to drug resistance, and they exist across many cell types and can export numerous classes of antibiotics. Cells with efflux pumps can regulate pump expression to maintain low intracellular drug concentrations. Here, we explored a three-dimensional evolutionary landscape, in which we mapped how resistance emerged depending on the antibiotic concentration, the presence of efflux pumps and their regulators, and time. We found that treatments just above the antibiotic concentration that inhibits growth of the parent strain were most likely to promote resistance, but that efflux pump levels influence the severity of these outcomes. These results indicate that there are specific treatment regimens and strain backgrounds that are especially problematic for the evolution of resistance.</jats:p></jats:sec>