Antibiotic susceptibility of <i>Escherichia coli</i> is affected by evolutionary history but not by history of elemental limitation

Antibiotic resistance in bacteria is a global public health threat. To understand how the evolution of antibiotic susceptibility is affected by environmental conditions and prior evolutionary history, we worked with populations from the Long-Term Evolution Experiment (LTEE) with <i>Escherichia coli</i>. These populations previously evolved independently for 50,000 generations in an environment without antibiotics, making them an ideal system for studying the effect of evolutionary history on adaptation to new selective pressures. We further evolved five of the LTEE populations, as well as their shared ancestor, under either carbon- or nitrogen-limited conditions and then tested intrinsic resistance to four antibiotics. Evolution under elemental limitation did not have a significant impact on resistance to any of the tested antibiotics. However, some LTEE populations did have higher resistance than other populations. Susceptibility also varied within one population, which had the lowest level of resistance to all four antibiotics. We hypothesized that resistance levels might differ between two clades of bacteria that have coexisted within this population for more than 40,000 generations. Interestingly, although antibiotic susceptibility varied within the population, there was no consistent difference between clades. Instead, one particular clone isolated from the population exhibited higher resistance than the other clones sampled. These findings indicate that antibiotic resistance can vary both within and between experimentally evolved populations, even in the absence of direct selection on resistance. Our results also show that measured levels of susceptibility may depend on stochastic sampling effects during isolation of clones.IMPORTANCEAntibiotic resistance is one of the most pressing health challenges worldwide, and understanding how bacteria evolve resistance, even when not directly exposed to antibiotics, is critical for managing and predicting emerging threats. Our study leverages the unique Long-Term Evolution Experiment with <i>Escherichia coli</i> to show that both the evolutionary history of bacterial populations and random variation among individual clones can significantly influence intrinsic antibiotic susceptibility. Our results also suggest that elemental limitation, while a critical environmental variable, may not be an important driver of intrinsic antibiotic susceptibility, at least over short time frames.