Amplification of a chromosomal beta-lactamase gene during amoxicillin resistance development in E. coli

Bacteria can acquire high levels of resistance after long-term exposure to sublethal concentrations of antimicrobials. Beta-lactams are the most used antimicrobials. The overexpression of the chromosomal beta-lactamase gene ampC, through gene amplification, is a crucial factor in beta-lactam resistance development in E. coli. This study focusses on this process of amplification and the factors influencing it. Development of resistance starts with mutations in the ampC promoter. The ampC amplification occurs about halfway during amoxicillin resistance evolution. When ampC is removed, no amplification is observed. When ampC is translocated, a DNA fragment around it at the new location is amplified. Replacing ampC by a tetracycline resistance gene and exposure to tetracycline yields amplification of a similar fragment. Of the genomic changes between the onset of ampC amplification and the end of the evolutionary process, mostly those located in sigma factor RpoD correlated with ampC amplification. These mutations, introduced as point mutations at the 445 and 570 amino acids of RpoD, enhance beta-lactam resistance. By removing some of the hitchhiking genes within the amplicons and the hotspot regions defining amplification boundaries, the sugE and epmA genes located neighbouring ampC were found to contribute to amoxicillin resistance development.