Stress tolerance studies are typically conducted in an all-or-none fashion. However, in realistic settings-such as in clinical or metabolic engineering applications-cells may encounter stresses at different rates. Therefore, how cells tolerate stress may depend on its rate of appearance. To address this, we studied how the rate of stress introduction affects bacterial stress tolerance by focusing on a key stress response mechanism. Efflux pumps, such as AcrAB-TolC of , are membrane transporters well known for the ability to export a wide variety of substrates, including antibiotics, signaling molecules, and biofuels. Although efflux pumps improve stress tolerance, pump overexpression can result in a substantial fitness cost to the cells. We hypothesized that the ideal pump expression level would involve a rate-dependent trade-off between the benefit of pumps and the cost of their expression. To test this, we evaluated the benefit of the AcrAB-TolC pump under different rates of stress introduction, including a step, a fast ramp, and a gradual ramp. Using two chemically diverse stresses, the antibiotic chloramphenicol and the jet biofuel precursor pinene, we assessed the benefit provided by the pumps. A mathematical model describing these effects predicted the benefit as a function of the rate of stress introduction. Our findings demonstrate that as the rate of introduction is lowered, stress response mechanisms provide a disproportionate benefit to pump-containing strains, allowing cells to survive beyond the original inhibitory concentrations. Efflux pumps are ubiquitous in nature and provide stress tolerance in the cells of species ranging from bacteria to mammals. Understanding how pumps provide tolerance has far-reaching implications for diverse fields, from medicine to biotechnology. Here, we investigated how the rate of stressor appearance impacts tolerance. We focused on two distinct substrates of AcrAB-TolC efflux pumps, the antibiotic chloramphenicol and the biofuel precursor pinene. Interestingly, tolerance is highly dependent on the rate of stress introduction. Therefore, it is important to consider not only the total quantity of a stressor but also the rate at which it is applied. The implications of this work are significant because environments are rarely static; antibiotic concentrations change during dosing, and metabolic engineering processes change with time.

Cell-cell interactions play an important role in bacterial antibiotic resistance. Here, we asked whether neighbor proximity is sufficient to generate single-cell variation in antibiotic resistance due to local differences in antibiotic concentrations. To test this, we focused on multidrug efflux pumps because recent studies have revealed that expression of pumps is heterogeneous across populations. Efflux pumps can export antibiotics, leading to elevated resistance relative to cells with low or no pump expression. In this study, we co-cultured cells with and without AcrAB-TolC pump expression and used single-cell time-lapse microscopy to quantify growth rate as a function of a cell’s neighbors. In inhibitory concentrations of chloramphenicol, we found that cells lacking functional efflux pumps (ΔacrB) grow more slowly when they are surrounded by cells with AcrAB-TolC pumps than when surrounded by ΔacrB cells. To help explain our experimental results, we developed an agent-based mathematical model, which demonstrates the impact of neighbors based on efflux efficiency. Our findings hold true for co-cultures of Escherichia coli with and without pump expression and also in co-cultures of E. coli and Salmonella typhumirium. These results show how drug export and local microenvironments play a key role in defining single-cell level antibiotic resistance.

Combatting the rise of antibiotic resistance is a significant challenge. Efflux pumps are an important contributor to drug resistance; they exist across many cell types and can export numerous classes of antibiotics.

ABSTRACTAntibiotic 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.IMPORTANCECombatting 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.