WCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.

Bacterial transglycosylation is an interesting target in antibiotic drug development. An in vitro transglycosylation assay was developed and used to search for possible inhibitors of Staphylococcus aureus Penicillin Binding Protein 2-mediated transglycosylation. Since the substrate, Lipid II, has no UV-chromophore, the assay relies on LC coupled to MS for analysis of the incubation mixtures. Extracts from Thymus sipyleus, Salvia verticillata, Salvia virgata and Oolong tea were tested, as well as epigallocatechin gallate and ursolic acid, which are chemical compounds derived from plants. Matrix effects hampered Lipid II quantification in samples treated with very high concentrations of extracts. None of these extracts or isolated compounds appeared to have inhibitory activities towards the transglycosylation function of Penicillin Binding Protein 2.