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

Ceftazidime-avibactam and cefiderocol are two of the latest generation beta-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales. Here, we show that structural changes in AmpC beta-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 beta-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.