Progression of <i>ampC</i> amplification during <i>de novo</i> amoxicillin resistance development in <i>E. coli</i>

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 <i>de novo</i> β-lactam resistance in <i>Escherichia coli</i>. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of <i>ampC</i> amplification and <i>ampC</i> promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. <i>AmpC</i> 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 <i>ampC</i> mRNA level by an average factor of 200 after 22 days. An IS<i>1</i> insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the <i>ampC</i> gene complementation strain (CompA), but not in ∆<i>ampC</i>, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and <i>ampC</i> amplification, the DNA mutations acquired during resistance evolution by the WT, ∆<i>ampC</i>, and CompA were analyzed. Compared to evolved ∆<i>ampC</i>, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆<i>ampC</i> did not show amplification of the fragment around the original <i>ampC</i> 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 <i>ampC</i> are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These <i>ampC</i>-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 <i>ampC</i> transcription enhancement, <i>ampC</i> amplification and <i>ampC</i> promoter mutations, during <i>de novo</i> amoxicillin resistance evolution. We propose that the transposon IS<i>1</i> contributes to the amplification <i>ampC</i> region, that the sigma factor 70 regulates <i>ampC</i> overexpression, and that these combined form the backbone of a putative mechanism for <i>ampC</i> amplification.