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

Abstract

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 arcA were also found to be associated with pJXP9 plasmid carriage among endpoint-evolved clones from diverse treatments. Meanwhile, arcA deletion improved the persistence of pJXP9 plasmid without drugs. Overall, our findings indicated that plasmid-borne MDR region deletion and chromosomal arcA inactivation mutation jointly contributed to co-adaptation and co-evolution between MDR IncHI2 plasmid and Salmonella Typhimurium under different drug selection pressure.