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 {beta}-lactam resistance in E. 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 in the evolution of resistance by stepwise increasing amoxicillin concentration. AmpC promoter mutations occur by day two, while the amplification by a factor of approximately eight occurs after more than six days of amoxicillin exposure. The combination of amplification and promoter mutations increase ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion was identified in the amplification junction, suggesting the amplification is facilitated by mobile genetic elements transposition. In order to identify the essential genes for ampC amplification, the chromosomal mutations of strains with induced amoxicillin resistance were compared a similarly evolved resistant {Delta}ampC knockout. The evolved {Delta}ampC contained several resistance mutations that were absent in the WT, which accumulated more mutations in stress response genes. The amoxicillin evolved {Delta}ampC does not show amplification of the fragment around the original ampC position but exhibits a large duplication or triplication at another position, suggesting selection of genes to amplify is essential for resistance adaption. IMPORTANCEAmoxicillin is one of the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin. These ampC amplification fragments have been identified in extended-spectrum beta-lactamases (ESBLs) plasmids, which are considered the main cause of beta- lactam resistance. Understanding the progression of ampC amplification enables amoxicillin resistance prevention. In this study, we show 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 and that the sigma factor 70 regulates ampC overexpression.