Experimental evolution of ceftazidime resistance in MG1655 with and without TEM-1 on a plasmid or inserted into the chromosome.. E. coli MG1655-TEM-1 ceftazidime resistance evolution

Plasmids are thought to play a key role in bacterial evolution by acting as vehicles for horizontal gene transfer, but the role of plasmids as catalysts of gene evolution remains unexplored. We challenged populations of E. coli carrying blaTEM-1 β-lactamase gene on either the chromosome or a multicopy plasmid (19/cell) with increasing concentrations of ceftazidime. The plasmid accelerated resistance evolution by increasing the rate of appearance of novel TEM-1 mutations conferring resistance to ceftazidime, and then by amplifying the effect of TEM-1 mutations due to increased gene dosage. Crucially, this dual effect was necessary and sufficient for the evolution of clinically relevant levels of resistance. Subsequent evolution occurred by mutations in a regulatory RNA that increased plasmid copy number, resulting in marginal gains in ceftazidime resistance. These results uncover a new role for multicopy plasmids as catalysts for the evolution of antibiotic resistance in bacteria.

质粒（plasmid）作为水平基因转移的载体，被认为在细菌进化中发挥关键作用，但质粒作为基因进化催化剂的功能仍未得到充分探索。本研究以浓度逐步升高的头孢他啶（ceftazidime）胁迫处理携带blaTEM-1 β-内酰胺酶（blaTEM-1 β-lactamase）基因的大肠杆菌（E. coli）种群，这些菌株的该基因分别整合于染色体（chromosome）或多拷贝质粒（multicopy plasmid，19拷贝/细胞）上。该质粒通过双重途径加速了抗药性进化：一是提升了可赋予头孢他啶抗药性的新型TEM-1突变的出现速率，二是通过提高基因剂量（gene dosage）增强TEM-1突变的效应。至关重要的是，这种双重效应对于细菌进化至临床相关水平的抗药性而言，既是必要条件也是充分条件。后续的进化则通过调控RNA（regulatory RNA）的突变实现，该突变提升了质粒拷贝数，使头孢他啶抗药性获得小幅提升。本研究结果揭示了多拷贝质粒作为细菌抗生素抗药性进化催化剂的全新功能。