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 (AmpCEnt385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpCEnt385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpCEnt385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpCEnt385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpCEnt385 and AmpCP99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpCEnt385 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.

Cefiderocol Activity Against ClinicalPseudomonas aeruginosaIsolates Exhibiting Ceftolozane-Tazobactam Resistance

“…These deletions appear to broadly impact cephalosporin antibiotics in that they confer resistance to cefepime, CAZ-AVI, and cefiderocol—in the absence of preceding exposure to CAZ-AVI or cefiderocol. The same group described a third patient with an E. cloacae clinical isolate with a cefiderocol MIC of >16 mcg/mL with an alanine–proline deletion at positions 294 and 295 and a leucine-to-valine substitution at position 296 in AmpC [ 10 ]. Conformation changes in the R2 loop of AmpC β-lactamases expand its substrate binding site, enabling entrapment of cephalosporins with bulkier R2 side chains, increasing their hydrolysis [ 24 ].…”

“…Before the clinical use of cefiderocol, there was widespread belief that resistance would primarily result from mutations in TonB-dependent receptors (TBDRs), a series of bacterial outer membrane proteins that mediate siderophore–iron complex transport [ 4-6 ]. While such mutations have been identified [ 7 , 8 ], there have also been isolated reports of changes in the ampC region contributing to cefiderocol resistance among the Enterobacterales [ 9 , 10 ]. This may occur after exposure to oxyminocephalosporins, such as CAZ-AVI or cefepime, in the absence of exposure to cefiderocol.…”

Structural Investigations of the Inhibition of Escherichia coli AmpC β-Lactamase by Diazabicyclooctanes

“…(v) Distances and angles. PDB entries were the following: AmpC Ent385 -AVI ( 55 ) ( 6LC8 ), PDC-1-AVI ( 29 ) ( 4OOY ), PDC-1-REL ( 20 ) ( 4NK3 ), TRU-1-AVI ( 28 ) ( 6FM7 ), PDC-1-NAC ( 18 ) ( 4X68 ), and FOX-1-AVI ( 27 ) ( 5ZA2 ). In the structure of the AmpC Ent385 -AVI complex, K67 is observed in two conformations; in one it is not positioned to interact with N152 ( 55 ).…”

Cefiderocol, a New Siderophore Cephalosporin for the Treatment of Complicated Urinary Tract Infections Caused by Multidrug-Resistant Pathogens: Preclinical and Clinical Pharmacokinetics, Pharmacodynamics, Efficacy and Safety

“…While neither resistance pattern to cefiderocol nor the underlying mechanisms have been studied, Kawai et al reported reduced susceptibility to cefiderocol in AmpC beta-lactamases with R2 loop deletion that increased hydrolysis of cefiderocol and ceftazidime-avibactam [ 19 ]. This finding explained the evolving survival strategy of MDR GNB to beta-lactam agents under selective pressure, and warrants further studies [ 19 ].…”