The global increase of antibiotic resistant bacteria threatens the modern health care and challenges the therapeutic effects of available antibiotics. The b-lactam mecillinam (Mec) is an exception to this due to a stable clinical resistance prevalence resistance of approximately 3%. It is only used to treat uncomplicated urinary tract infections (UTIs), mainly caused by E. coli. Mecillinam resistance (MecR) is easily selected for in laboratory settings and linked to >40 genes, including the mrdA gene encoding the Mec target penicillin-binding protein 2. A majority of the known MecR mutations confer a severe fitness cost. Fitness is important for bacteria to survive in the bladder and clinical isolates have been shown to have high fitness. These isolates contain loss-of-function mutations in the cysB gene, which encode a positive regulator of cysteine biosynthesis. In a previous evolution experiment, fitness cost of cysB and mrdA MecR mutations was compensated and the compensatory mutations were identified. Here the compensatory mutations were reconstructed into wildtype (WT) E. coli strain MG1655, and cysB and mrdA backgrounds to study the impact of the mutations on resistance and fitness, using MIC tests and Bioscreen C assays. Our results show that the mrdA mutants only had partial fitness compensation (significantly lower fitness than WT) for all strains and all strains also increased their MecR. The low fitness is possibly an explanation for the lack of mrdA mutants outside laboratories. Of the clinically relevant cysB mutants the majority lost their resistance when increasing growth rate, some even to levels significantly higher than WT, indicating that DcysB mutations are easier to compensate for. One strain (ydjNmx2) however, had a significantly higher growth rate while remaining clinically MecR.