Characterization of the intrinsic AmpC β-lactamase of Escherichia marmotae and its impact on decreased susceptibility to cefiderocol and resistance to broad-spectrum cephalosporins.

OBJECTIVES

We aimed to characterize the intrinsic AmpC β-lactamase (EMC-2) from an Escherichia marmotae clinical isolate recovered from a bloodstream infection, exhibiting resistance to broad-spectrum cephalosporins.

METHODS

Cloning experiments in Escherichia coli were performed to evaluate the impact of the respective AmpC enzymes on β-lactam susceptibility. Purification of the enzymes was performed by ion-exchange chromatography, and kinetic assays were performed by UV spectrophotometry.

RESULTS

By comparing the sequence of this AmpC with that of a WT E. marmotae, four amino acid substitutions and one amino acid deletion at position 296 were identified. Furthermore, a promoter alteration was responsible for an enhanced gene expression. Production of EMC-2 in E. coli TOP10 conferred an extended-spectrum AmpC (ESAC) phenotype characterized by increased MICs of cefotaxime, cefepime and cefiderocol. Site-directed mutagenesis confirmed the important role of the Arg296 deletion in this phenotype.

CONCLUSIONS

We showed that the intrinsic AmpC of E. marmotae can acquire both structural and regulatory modifications that broaden its substrate spectrum, leading to reduced susceptibility to clinically relevant β-lactams, including cefiderocol.