Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing ␤-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug-and strain-specific patterns.