Effects of a previously selected antibiotic resistance on mutations acquired during development of a second resistance in Escherichia coli

BACKGROUND: The effect of mutations conferring antibiotic resistance can depend on the genetic background. To determine if a previously de novo acquired antibiotic resistance influences the adaptation to a second antibiotic, antibiotic resistance was selected for by exposure to stepwise increasing sublethal levels of amoxicillin, enrofloxacin, kanamycin, or tetracycline. E. coli populations adapted to either a single or two antibiotics sequentially were characterized using whole genome population sequencing and MIC measurements. RESULTS: In a wild-type background, adaptation to any of the antibiotics resulted in the appearance of well-known mutations, as well as a number of mutated genes not known to be associated with antibiotic resistance. Development of a second resistance in a strain with an earlier acquired resistance to a different antibiotic did not always result in the appearance of all mutations associated with resistance in a wild-type background. In general, a more varied set of mutations was acquired during secondary adaptation. The ability of E. coli to maintain the first resistance during this process depended on the combination of antibiotics used. The maintenance of mutations associated with resistance to the first antibiotic did not always predict the residual MIC for that compound. CONCLUSIONS: In general, the data presented here indicate that adaptation to each antibiotic is unique and independent. The mutational trajectories available in already resistant cells appear more varied than in wild-type cells, indicating that the genetic background of E. coli influences resistance development. The observed mutations cannot always fully explain the resistance pattern observed, indicating a crucial role for adaptation on the gene expression level in de novo acquisition of antibiotic resistance.