Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in Enterobacter cloacae Due to AmpC R2 Loop Deletion

Abstract

Ceftazidime-avibactam and cefiderocol are two of the latest generation β-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales Here, we show that structural changes in AmpC β-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime-avibactam and cefiderocol without prior exposure to either agent. The AmpC β-lactamase of Ent385 (AmpC^Ent385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpC^Ent385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpC^Ent385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpC^Ent385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpC^Ent385 and AmpC^P99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpC^Ent385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.

Keywords: avibactam; beta-lactamase; cefiderocol; ceftazidime.

FIG 1

Chemical structures of avibactam (a), ceftazidime (b), and cefiderocol (c). The R1 and R2 side chains of cephalosporin are labeled. This figure was prepared by using MarvinSketch 19.20 (2019; ChemAxon).

FIG 2

Overall structures of AmpC^Ent385. (a) The AmpC^Ent385 structures are shown as cartoon representations and colored white for the AmpC^Ent385 free form, blue for the AmpC^Ent385-CAZ complex, and cyan for the AmpC^Ent385-AVI complex. To indicate the active site, the ceftazidime molecule is shown as CPK (space-filling model) representation colored magenta. (b and c) 2mF_o-DF_c maps are shown as green mesh contoured 1.5σ. The molecules of ceftazidime and avibactam are shown as ball-and-stick representations colored magenta and pink, respectively.

FIG 3

Structure comparison of AmpC^Ent385 and AmpC^P99. The structures of AmpC^Ent385 and AmpC^P99 are shown as cartoon representations and colored white and gray, respectively. To clarify the difference, the colors of the V283–V294 residue structures in AmpC^Ent385 and the corresponding residues in AmpC^P99 appear in blue and green, respectively.

FIG 4

Ceftazidime recognition by AmpC^Ent385. (a) Stereo view of the ceftazidime binding. The ceftazidime molecule as shown in ball-and-stick representations colored magenta. Hydrogen bonds are shown as orange dashed lines. Residues and the water molecules participating the hydrogen bond network are shown as white stick and red spheres, respectively. (b) The deacylating water molecule is shown as a large red sphere. Red dashed line indicates the distance between C-8 atom of ceftazidime and the deacylating water molecule. (c and d) Comparison of the substrate-binding sites of the AmpC^Ent385-CAZ complex and the AmpC^Ec-CAZ complex. Transparent molecular surfaces of AmpC^Ent385 (c) and AmpC^Ec (d) are colored white and gray, respectively. To clarify the difference, the colors of the V283–V294 residue structures, including R2 loop in AmpC^Ent385, and corresponding residues in AmpC^P99 appear in blue and green, respectively. Arrowheads indicate the R2 side chain positions of ceftazidime.

FIG 5

Avibactam recognition by AmpC^Ent385. (a) Stereo view of the avibactam binding. The avibactam molecule is shown as ball-and-stick representations colored pink. Hydrogen bonds are shown as orange dashed lines. Residues and the water molecules participating the hydrogen bond network are shown as white stick and red spheres, respectively. (b) Structure comparisons of the AmpC^Ent385-AVI complex to the other class C β-lactamase complex with avibactam. Colors are white for AmpC^E385, purple for PDC-1 (PDB ID 4OOY), green for FOX-4 (PDB ID 5ZA2), and yellow for TRU-1 (PDB ID 6FM7). Red dashed line indicates a unique hydrogen bond between N289 of AmpC^Ent385 and the sulfate group of avibactam. Residues of AmpC^Ent385 are labeled. (c and d) Comparison of the substrate-binding sites of the AmpC^Ent385-AVI complex and the PDC-1–AVI complex (PDB ID 4OOY). Transparent molecular surfaces of AmpC^Ent385 (c) and PDC-1 (d) are colored in white and gray, respectively. To clarify the difference, the colors of the V283–V294 residue structures, including the R2 loop in AmpC^Ent385, and corresponding residues in PDC-1 appear in cyan and pale green, respectively. Arrowheads indicate the sulfate group of avibactam.