Mutation in the two-component regulator BaeSR mediates cefiderocol resistance and enhances virulence in .

has become one of the most challenging pathogens in many countries with limited treatment options available. Cefiderocol, a novel siderophore-conjugated cephalosporin, shows potent activity against , including isolates resistant to carbapenems. To date, few reports on the mechanisms of cefiderocol resistance are available. In order to investigate potential mechanisms of cefiderocol resistance in , we performed evolution experiments at sub-lethal concentrations of the antibiotic. All four cefiderocol-resistant strains obtained harbored mutations in two-component system BaeS-BaeR. When we engineered the mutations of BaeS (D89V) and BaeR (S104N) into the genome of ATCC 17978, these mutations increased cefiderocol minimum inhibitory concentrations (MICs) by 8-fold to 16-fold. Transcriptome analyses showed that the expression of MacAB-TolC and MFS transporters was up-regulated in BaeSR mutants. Strains over-expressing MFS transporter and MacAB-TolC displayed higher MICs and higher median inhibition concentration (IC) values, while MICs and IC decreased when efflux pump genes were knocked out. In a BaeR mutant with up-regulated operon, we observed a higher number of pili, enhanced surface motility, and increased biofilm formation compared to wild-type ATCC 17978. Using the infection model, we found that the BaeS mutant in which operon was up-regulated exhibited increased virulence. In conclusion, the mutations in BaeSR decreased cefiderocol susceptibility of through up-regulating efflux pumps gene expression. BaeS or BaeR also controls the expression of and , influencing biofilm formation, surface motility, and virulence in . IMPORTANCE The widespread prevalence of ulti-rugesistant (MDRAB) poses a significant therapeutic challenge. Cefiderocol is considered a promising antibiotic for the treatment of MDRAB infections. Therefore, it is necessary to study the potential resistance mechanisms of cefiderocol to delay the development of bacterial resistance. Here, we demonstrated that mutations in and reduced the susceptibility of to cefiderocol by up-regulating the expression of the MFS family efflux pump and MacAB-TolC efflux pump. We propose that BaeS mutants increase bacterial virulence by up-regulating the expression of the operon. This also reports the regulatory effect of BaeSR on operon for the first time. This study provides further insights into the role of BaeSR in developing cefiderocol resistance and virulence in .