Driving factors for beta-lactam resistance gene amplification during de novo resistance evolution in E. coli

Abstract

Long-term exposure of E. coli to non-lethal step-wise increasing concentrations of beta-lactam antibiotics induces high levels of resistance that can be accompanied by amplification of a chromosomal fragment around the ampC gene. We compared the amplification of the ampC fragment in the wildtype, an ampC knockout mutant, a mutant in which the ampC gene was replaced by a tetracycline resistance gene tet(B)and a strain in which the ampC has been translocated. When ampC was removed, no amplification occurred at the original ampC location, but DNA fragments were amplified around the genes coding for efflux pump AcrAB and the multiple antibiotic resistance operon MarRAB. When tet(B) replaced ampC, exposure to tetracycline induced amplification of comparable fragments, while exposure to amoxicillin induced duplication of a larger fragment elsewhere. When ampC was translocated, a fragment around it at the new location was amplified. The importance of the presence but not of the location within the chromosome of the resistance genes for the amplification process indicates that the mechanisms are neither gene nor location specific. Without the relatively efficient resistance gene ampC, duplication and amplification occur around acrAB and marRAB that code for amoxicillin and tetracycline resistance factors. These duplications and amplifications are prevented by ampC amplification.