Phenotype evaluation rawdata of Acinetobacter baumannii harboring chromosomal parallel mutations or an evolved plasmid

OXA-23 is the predominant carbapenemase in carbapenem-resistant
Acinetobacter baumannnii. The co-evolutionary dynamics of A. baumannii and
OXA-23-encoding plasmids are poorly understood. Here, we transformed A.
baumannnii ATCC 17978 with pAZJ221, a blaOXA-23-containing plasmid from a
clinical A. baumannnii 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
blaOXA-23 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 causal effects of these mutations
on fitness costs and plasmid kinetics, the evolved plasmid containing two
copies of blaOXA-23 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 blaOXA-23 contributed to host
fitness under carbapenem pressure but imposed a burden on the host in
antibiotic-free media relative to the unevolved pAZJ221. Overall, our
study provides a framework for the co-evolution of A. baumannii and a
clinical blaOXA-23-containing plasmid, involving early blaOXA-23
duplication followed by chromosomal adaptations.