Zidebactam and WCK 5153 are novel beta-lactam enhancers that are bicyclo-acyl hydrazides (BCH), derivatives of the diazabicyclooctane (DBO) scaffold, targeted for the treatment of serious infections caused by highly drug-resistant Gram-negative pathogens. In this study, we determined the penicillin-binding protein (PBP) inhibition profiles and the antimicrobial activities of zidebactam and WCK 5153 against Pseudomonas aeruginosa, including multidrug-resistant (MDR) metallo- beta-lactamase (MBL)-producing high-risk clones. MIC determinations and time-kill assays were conducted for zidebactam, WCK 5153, and antipseudomonal beta-lactams using wild-type PAO1, MexAB-OprM-hyperproducing (mexR), porin-deficient (oprD), and AmpC-hyperproducing (dacB) derivatives of PAO1, and MBL-expressing clinical strains ST175 (bla(VIM-2)) and ST111 (bla(VIM-1)). Furthermore, steady-state kinetics was used to assess the inhibitory potential of these compounds against the purified VIM-2 MBL. Zidebactam and WCK 5153 showed specific PBP2 inhibition and did not inhibit VIM-2 (apparent K-i [K-i app] > 100 mu M). MICs for zidebactam and WCK 5153 ranged from 2 to 32 mu g/ml (amdinocillin MICs > 32 mu g/ml). Time-kill assays revealed bactericidal activity of zidebactam and WCK 5153. LIVE-DEAD staining further supported the bactericidal activity of both compounds, showing spheroplast formation. Fixed concentrations (4 or 8 mu g/ml) of zidebactam and WCK 5153 restored susceptibility to all of the tested beta-lactams for each of the P. aeruginosa mutant strains. Likewise, antipseudomonal beta-lactams (CLSI breakpoints), in combination with 4 or 8 mu g/ml of zidebactam or WCK 5153, resulted in enhanced killing. Certain combinations determined full bacterial eradication, even with MDR MBL-producing high-risk clones. beta-Lactam-WCK enhancer combinations represent a promising beta-lactam "enhancer-based" approach to treat MDR P. aeruginosa infections, bypassing the need for MBL inhibition.