Co-evolutionary adaptations of and a clinical carbapenemase-encoding plasmid during carbapenem exposure.

OXA-23 is the predominant carbapenemase in carbapenem-resistant . The co-evolutionary dynamics of and OXA-23-encoding plasmids are poorly understood. Here, we transformed ATCC 17978 with pAZJ221, a -containing plasmid from clinical isolate A221, and subjected the transformant to experimental evolution in the presence of a sub-inhibitory concentration of imipenem for nearly 400 generations. We used population sequencing to track genetic changes at six time points and evaluated phenotypic changes. Increased fitness of evolving populations, temporary duplication of in pAZJ221, interfering allele dynamics, and chromosomal locus-level parallelism were observed. To characterize genotype-to-phenotype associations, we focused on six mutations in parallel targets predicted to affect small RNAs and a cyclic dimeric (3' → 5') GMP-metabolizing protein. Six isogenic mutants with or without pAZJ221 were engineered to test for the effects of these mutations on fitness costs and plasmid kinetics, and the evolved plasmid containing two copies of was transferred to ancestral ATCC 17978. Five of the six mutations contributed to improved fitness in the presence of pAZJ221 under imipenem pressure, and all but one of them impaired plasmid conjugation ability. The duplication of increased host fitness under carbapenem pressure but imposed a burden on the host in antibiotic-free media relative to the ancestral pAZJ221. Overall, our study provides a framework for the co-evolution of and a clinical -containing plasmid in the presence of imipenem, involving early duplication followed by chromosomal adaptations that improved the fitness of plasmid-carrying cells.