Progression of ampC amplification during de novo amoxicillin resistance development in E. coli

Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in Escherichia coli. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of ampC amplification and ampC promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. AmpC promoter mutations occurred by day 2, while the approximately eight-fold amplification occurred after more than 6 days of amoxicillin exposure. The combination of the amplification and the promoter mutations increased the ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the ampC gene complementation strain (CompA), but not in ∆ampC, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and ampC amplification, the DNA mutations acquired during resistance evolution by the WT, ∆ampC, and CompA were analyzed. Compared to evolved ∆ampC, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆ampC did not show amplification of the fragment around the original ampC position but exhibited a large duplication or triplication at another position, suggesting the essential role of the duplicated genes in resistance development.IMPORTANCEAmoxicillin is the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These ampC-containing fragments have been identified in extended-spectrum beta-lactamase plasmids, which are considered the main cause of beta-lactam resistance. In this study, we document the time course of two important factors for ampC transcription enhancement, ampC amplification and ampC promoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification ampC region, that the sigma factor 70 regulates ampC overexpression, and that these combined form the backbone of a putative mechanism for ampC amplification.