Low levels of tetracyclines select for a mutation that prevents the evolution of high-level resistance to tigecycline

In a collection of Escherichia coli isolates, we discovered a new mechanism leading to frequent and high-level tigecycline resistance involving tandem gene amplifications of an efflux pump encoded by the tet(A) determinant. Some isolates, despite carrying a functional tet(A), could not evolve high-level tigecycline resistance by amplification due to the presence of a deletion in the TetR(A) repressor. This mutation impaired induction of tetA(A) (encoding the TetA(A) efflux pump) in presence of tetracyclines, with the strongest effect observed for tigecycline, subsequently preventing the development of tet(A) amplification-dependent high-level tigecycline resistance. We found that this mutated tet(A) determinant was common among tet(A)-carrying E. coli isolates and analysed possible explanations for this high frequency. First, while the mutated tet(A) was found in several ST-groups, we found evidence of clonal spread among ST131 isolates, which increases its frequency within E. coli databases. Second, evolution and competition experiments revealed that the mutation in tetR(A) could be positively selected over the wild-type allele at sub-inhibitory concentrations of tetracyclines. Our work demonstrates how low concentrations of tetracyclines, such as those found in contaminated environments, can enrich and select for a mutation that generates an evolutionary dead-end that precludes the evolution towards high-level, clinically relevant tigecycline resistance.