Evolved E.coli K12 populations with streptomycin resistance selected by pesticides and low-level streptomycin

Identifying stressors that can influence antibiotic resistance evolution is pivotal to understand and control the development and widespread of resistant bacteria in many environments. Antibiotics are found to select for resistant mutations and shape evolutionary pathways. However, how co-occurring environmental stressors influence evolutionary trajectories toward antibiotic resistance remains unknown. To explore this, we exposed susceptible Escherichia coli K-12 populations to low-level streptomycin together with environmental-level pesticides as the co-stressor and evolved for 500 generations. Phenotypic resistance development of the evolved populations was determined, and genetic mutations corresponding to the phenotypic resistance development were identified by whole-genome and whole-population sequencing. Our results showed that low-level streptomycin itself could not induce strong resistance in the populations. However, the addition of pesticides can drive the evolutionary paths to strong resistance under low-level streptomycin selection. Unique molecular mechanisms responsible for resistance were identified in the populations under the selection with pesticides, such as mutations related to target modification and oxidative stress defense (rpsL and dsbC mutations). The mutational trajectories under the selection with pesticides showed sequential fixation of mildly and strongly resistant mutants in the populations, which was driven by the fitness disparity of these mutants. We further demonstrated that the drastically elevated resistance of the populations could be caused by the presence of a small number of strongly resistant mutants, even less than 1%. Together, these findings demonstrated the effect of pesticides as a co-stressor stimulating the emergence of resistant mutants and drive the evolutionary path toward strong resistance under low-level antibiotic selection.