Zidebactam and WCK 5153 are novel ␤-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-␤-lactamase (MBL)-producing high-risk clones. MIC determinations and time-kill assays were conducted for zidebactam, WCK 5153, and antipseudomonal ␤-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 ] Ͼ 100 M). MICs for zidebactam and WCK 5153 ranged from 2 to 32 g/ml (amdinocillin MICs Ͼ 32 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 g/ml) of zidebactam and WCK 5153 restored susceptibility to all of the tested ␤-lactams for each of the P. aeruginosa mutant strains. Likewise, antipseudomonal ␤-lactams (CLSI breakpoints), in combination with 4 or 8 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. ␤-Lactam-WCK enhancer combinations represent a promising ␤-lactam "enhancerbased" approach to treat MDR P. aeruginosa infections, bypassing the need for MBL inhibition.

The clinical and epidemiological threat of the growing antimicrobial resistance in Gram-negative pathogens, particularly for β-lactams, the most frequently used and relevant antibiotics, urges research to find new therapeutic weapons to combat the infections caused by these microorganisms. An essential previous step in the development of these therapeutic solutions is to identify their potential targets in the biology of the pathogen. This is precisely what we sought to do in this review specifically regarding the barely exploited field analyzing the interplay among the biology of the peptidoglycan and related processes, such as β-lactamase regulation and virulence. Hence, here we gather, analyze, and integrate the knowledge derived from published works that provide information on the topic, starting with those dealing with the historically neglected essential role of the Gram-negative peptidoglycan in virulence, including structural, biogenesis, remodeling, and recycling aspects, in addition to proinflammatory and other interactions with the host. We also review the complex link between intrinsic β-lactamase production and peptidoglycan metabolism, as well as the biological costs potentially associated with the expression of horizontally acquired β-lactamases. Finally, we analyze the existing evidence from multiple perspectives to provide useful clues for identifying targets enabling the future development of therapeutic options attacking the peptidoglycan-virulence interconnection as a key weak point of the Gram-negative pathogens to be used, if not to kill the bacteria, to mitigate their capacity to produce severe infections.

Multidrug-resistant has rapidly spread worldwide, resulting in a serious threat to hospitalized patients. Zidebactam and WCK 5153 are novel non-β-lactam bicyclo-acyl hydrazide β-lactam enhancer antibiotics being developed to target multidrug-resistant The objectives of this work were to determine the 50% inhibitory concentrations (ICs) for penicillin-binding proteins (PBP), the OXA-23 inhibition profiles, and the antimicrobial activities of zidebactam and WCK 5153, alone and in combination with β-lactams, against multidrug-resistant MICs and time-kill kinetics were determined for an clinical strain producing the carbapenemase OXA-23 and belonging to the widespread European clone II of sequence type 2 (ST2). Inhibition of the purified OXA-23 enzyme by zidebactam, WCK 5153, and comparators was assessed. All of the compounds tested displayed apparent values of>100 μM, indicating poor OXA-23 β-lactamase inhibition. The ICs of zidebactam, WCK 5153, cefepime, ceftazidime, meropenem, and sulbactam (range of concentrations tested, 0.02 to 2 μg/ml) for PBP were also determined. Zidebactam and WCK 5153 demonstrated specific high-affinity binding to PBP2 of (0.01 μg/ml for both of the compounds). The MICs of zidebactam and WCK 5153 were>1,024 μg/ml for wild-type and multidrug-resistant strains. Importantly, combinations of cefepime with 8 μg/ml of zidebactam or WCK 5153 and sulbactam with 8 μg/ml of zidebactam or WCK 5153 led to 4- and 8-fold reductions of the MICs, respectively, and showed enhanced killing. Notably, several of the combinations resulted in full bacterial eradication at 24 h. We conclude that zidebactam and WCK 5153 are PBP2 inhibitors that show a potent β-lactam enhancer effect against, including a multidrug-resistant OXA-23-producing ST2 international clone.

Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers’ interest as a source of therapeutic targets.