Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in <i>Enterobacter cloacae</i> 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 <i>Enterobacterales</i> Here, we show that structural changes in AmpC β-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant <i>Enterobacter cloacae</i> 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 <i>Escherichia coli</i> 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 <i>E. cloacae</i> 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 <i>ampC</i> to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.