ABSTRACTFosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused byEscherichia coli. In recent years, quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found thatackAandptacontribute to fosfomycin activity againstE. coli.ackAandptamutantE. colihad reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition,ackAandptamutants had decreased expression ofglpTthat encodes one of the fosfomycin transporters. Expression ofglpTis enhanced by a nucleoid-associated protein, Fis. We found that mutations inackAandptaalso caused a decrease infisexpression. Thus, we interpret the decrease inglpTexpression inackAandptadefective strains to be due to a decrease in Fis levels in these mutants. Furthermore,ackAandptaare conserved in multidrug-resistantE. coliisolated from patients with pyelonephritis and enterohemorrhagicE. coli, and deletion ofackAandptafrom these strains resulted in decreased susceptibility to fosfomycin. These results suggest thatackAandptainE. colicontribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin.IMPORTANCEThe spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of theackAandptagenes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.