Antibiotic resistance is a worldwide health threat, as bacteria continue to evade antibiotic treatment. In order to survive, bacteria utilize a number of resistance mechanisms, including efflux pumps, which efficiently export antibiotics outside of the cell to reduce intracellular damage. While such mechanisms are well known, there remains a significant gap in knowledge regarding how different environmental dynamics, such as the rate of antibiotic introduction or the diversity within a microbial community, play a role in resistance. In this work, we used the AcrAB-TolC efflux pump as a case study to explore how such complex dynamics promote antibiotic resistance and its evolution. First, through a combined effort using experiments and mathematical modeling, we discovered that the rate of antibiotic introduction impacts the fraction of resistant bacteria in a population. We then explored the impact of mixed populations on survival following antibiotic treatment. In mixed microcolonies, we found that resistant cells can harm their susceptible neighbors by exporting antibiotics to increase the local concentrations of these drugs. Next, we aimed to understand how these environmental effects may impact longer-term survival of an antibiotic treatment, focusing on the evolution of resistance over ~72 hours. Through a series of adaptive evolution experiments, we identified that near-MIC treatments were the most likely to promote antibiotic resistance, regardless of whether the strains contained the AcrAB-TolC pump at wild type or overexpressed levels, or whether the strains lacked the pump altogether. In studying antibiotic introduction rates on evolution, we found that slower introduction rates facilitated the evolution of high levels of resistance with a minimal fitness cost. Meanwhile, mixed populations demonstrated limited evolvability after rapid antibiotic introductions. This work provides important insights into the impacts of environmental factors, such as the rate of antibiotic introduction and the homogeneity of populations, on the promotion and evolution of antibiotic resistance. These lessons may help inform future policies on antibiotic use and mitigate the continued pattern of resistance evolution
