Fitness effect of plasmid acquisition and experimental procedures. (a) An ExPEC strain (black) acquired each of the two MDR plasmids pG06-VIM-1 (green; 53 kB; IncR; Di Luca et al. 2017) and pK71-77-1-NDM (purple; 145 kB; IncC; Gama et al. 2020) of clinical origin encoding the carbapenemases VIM-1 and NDM-1, respectively. (b) Initial fitness costs of newly transferred plasmids in strains ExPEC+VIM and ExPEC+NDM (n = 4 and 3, respectively). Significant plasmid costs are indicated by asterisks (P = * < 0.05, ** < 0.01, *** < 0.001; one-sample t-test, two-sided). Error bars indicate ±SEM. (c) Experimental evolution in absence of selective pressure (∼300 generations) resulted in plasmid-carrying (Pop 1–4_VIM and Pop 5–8_NDM) and plasmid-free (Pop 9–12) populations which were subjected to whole-genome sequencing (WGS). Representative clones per plasmid-carrying evolved population (Clones 1–4_VIM and Clones 5–8_NDM) were sequenced and segregants without evolved pG06-VIM-1 (filled green circle) were generated for subsequent competition experiments (Clones 1–4).