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.