Genetic and biochemical characterization of OXA-1054, a carbapenem-hydrolyzing class D β-lactamase conferring broad-spectrum β-lactam resistance in Pseudomonas aeruginosa.

We aimed to characterize OXA-1054, a novel carbapenem-hydrolyzing class D β-lactamase (CHDL) detected in a multidrug-resistant Pseudomonas aeruginosa clinical isolate. Antimicrobial susceptibility was determined by broth microdilution, and carbapenemase activity was confirmed by hydrolysis assays. Whole-genome sequencing via Illumina and PacBio platforms enabled comprehensive analysis of the resistome and plasmid architecture. The blaOXA-1054 gene was cloned in parallel with blaOXA-48 and blaOXA-198 into the pUCP24 plasmid. β-Lactamases were produced in P. aeruginosa PAO1 for comparative evaluation of the phenotypic impact of each. OXA-48, OXA-198, and OXA-1054 β-lactamases were purified and further subjected to steady-state kinetic analysis, and 50% inhibitory activity of β-lactamase inhibitors was determined. The clinical P. aeruginosa isolate ARGA00461 showed resistance to carbapenems and most β-lactam/β-lactamase inhibitor combinations and tested positive in carbapenem hydrolysis assays. Genomic analysis revealed that the P. aeruginosa isolate ARGA00461 carried a gene coding for a previously uncharacterized CHDL, designated OXA-1054, which is closely related to the OXA-372 enzyme of environmental origin. The blaOXA-1054 gene was located in a small (≈5 kbp) non-conjugative plasmid coexisting with a conjugative IncP plasmid, which likely facilitated its mobilization. Production of OXA-1054 in P. aeruginosa PAO1 conferred a broader spectrum of β-lactam resistance than other CHDLs. Enzyme kinetics confirmed carbapenem hydrolysis with catalytic efficiency comparable to OXA-48 and revealed higher affinity for cefepime compared to OXA-48 and OXA-198. Among the inhibitors tested, only avibactam demonstrated relevant inhibitory activity. OXA-1054 mediates broad-spectrum β-lactam resistance in P. aeruginosa, including carbapenems and β-lactam/β-lactamase inhibitor combinations.