Evolution and maintenance of a large multidrug-resistant plasmid in a <i>Salmonella enterica</i> Typhimurium host under differing antibiotic selection pressures

The dissemination of antibiotic resistance genes (ARGs) through plasmids is a major mechanism for the development of bacterial antimicrobial resistance. The adaptation and evolution mechanisms of multidrug-resistant (MDR) plasmids with their hosts are not fully understood. Herein, we conducted experimental evolution of a 244 kb MDR plasmid (pJXP9) under various conditions including no antibiotics and mono- or combinational drug treatments of colistin (CS), cefotaxime (CTX), and ciprofloxacin (CIP). Our results showed that long-term with or without positive selections for pJXP9, spanning approximately 600 generations, led to modifications of the plasmid-encoded MDR and conjugative transfer regions. These modifications could mitigate the fitness cost of plasmid carriage and enhance plasmid maintenance. The extent of plasmid modifications and the evolution of plasmid-encoded antibiotic resistance depended on treatment type, particularly the drug class and duration of exposure. Interestingly, prolonged exposure to mono- and combinational drugs of CS and CIP resulted in a substantial loss of the plasmid-encoded MDR region and antibiotic resistance, comparable to the selection condition without antibiotic. By contrast, combinational treatment with CTX contributed to the maintenance of the MDR region over a long period of time. Furthermore, drug selection was able to maintain and even amplify the corresponding plasmid-encoded ARGs, with co-selection of ARGs in the adjacent regions. In addition, parallel mutations in chromosomal <i>arcA</i> were also found to be associated with pJXP9 plasmid carriage among endpoint-evolved clones from diverse treatments. Meanwhile, <i>arcA</i> deletion improved the persistence of pJXP9 plasmid without drugs. Overall, our findings indicated that plasmid-borne MDR region deletion and chromosomal <i>arcA</i> inactivation mutation jointly contributed to co-adaptation and co-evolution between MDR IncHI2 plasmid and <i>Salmonella</i> Typhimurium under different drug selection pressure.IMPORTANCEThe plasmid-mediated dissemination of antibiotic resistance genes has become a significant concern for human health, even though the carriage of multidrug-resistant (MDR) plasmids is frequently associated with fitness costs for the bacterial host. However, the mechanisms by which MDR plasmids and bacterial pairs evolve plasmid-mediated antibiotic resistance in the presence of antibiotic selections are not fully understood. Herein, we conducted an experimental evolution of a large multidrug-resistant plasmid in a <i>Salmonella enterica</i> Typhimurium host under single and combinatorial drug selection pressures. Our results show the adaptive evolution of plasmid-encoded antibiotic resistance through alterations of the MDR region in the plasmid, in particular substantial loss of the MDR region, in response to different positive selections, especially mono- and combinational drugs of colistin and ciprofloxacin. In addition, strong parallel mutations in chromosomal <i>arcA</i> were associated with pJXP9 carriage in <i>Salmonella</i> Typhimurium from diverse treatments. Our results thus highlight promoting the loss of the plasmid's MDR region could offer an alternative approach for combating plasmid-encoded antibiotic resistance.