Pseudomonas aeruginosa is an opportunistic pathogen that causes considerable morbidity and mortality, specifically in the intensive care. Antibiotic resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory, P. aeruginosa rapidly developed resistance against five medically relevant antibiotics upon exposure to step-wise increasing concentrations. At several time points during the acquisition of resistance samples were taken for whole genome sequencing. The increase of MIC for ciprofloxacin was linked to specific mutations in gyrA, parC and gyrB, appearing sequentially. In the case of tobramycin, mutations were induced in fusA, HP02880, rplB and capD The MIC for the beta-lactam compounds meropenem, ceftazidime and the combination piperacillin/tazobactam correlated linearly with the beta-lactamase activity, but not always with individual mutations. The genes that were mutated during development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reverted, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions, or somewhat lower growth rate. In all cases except ciprofloxacin, the increase of resistance seems to be the result of a complex interaction between several cellular systems, rather than individual mutations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.