Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9

Abstract

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the ST^Pas25 (international clone IC7), was highly resistant to β-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a bla_NDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the bla_NDM-1 or bla_NDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (K_m = 53 μM) than for NDM-1 (K_m = 161 μM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 μM^-1.s^-1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol.

Keywords: Acinetobacter baumannii; NDM; carbapenemases; cefiderocol; resistance.

Figure 1

Linear sequence alignment of plasmids pNDM-9, pIOMTU433 (accession number: AP014650) and pPM194122 (accession number: CP050426). Yellow arrows indicate the open reading frames of pNDM-9 plasmid; blue arrows indicate the Tn125 transposon and pink arrow indicates the bla_NDM-9 gene.

Figure 2

(A,C) Docking conformation of cefiderocol (represented as green sticks) in the active site of NDM-9 (represented as cyan cartoon and surface, respectively). (B,D) The same docking conformation superposed with the active site of NDM-1 (represented as pink cartoon and surface, respectively). The black ellipse highlights the repulsive interaction between the side chain of Glu152 and the carboxylate group from the R1 substituent in cefiderocol. Hydrogen bonds are represented as black springs.