González-Pinto, LucíaMonge-Olivares, LauraPérez-Rodríguez, GloriaAja-Macaya, PabloSánchez-Peña, LucíaGomis-Font, María AntoniaRodríguez-Pallares, SaludBlanco Martín, TaniaLópez-Cerero, LorenaBeceiro Casas, AlejandroBogaerts, PierreOliver, AntonioBou, GermánArca-Suárez, Jorge2026-08-202026-08-202026-05-29González-Pinto L, Monge-Olivares L, Pérez-Rodríguez G, Aja-Macaya P, Sánchez-Peña L, Gomis-Font MA, Rodríguez-Pallares S, Blanco-Martín T, López-Cerero L, Beceiro A, Bogaerts P, Oliver A, Bou G, Arca-Suárez J. Genetic and biochemical characterization of OXA-1054, a carbapenem-hydrolyzing class D β-lactamase conferring broad-spectrum β-lactam resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2026 Jul;70(7):e0180525.1098-6596https://hdl.handle.net/2183/49058[Abstract] 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.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/Carbapenem resistanceCarbapenem-resistant Pseudomonas aeruginosaCarbapenemase-producing Pseudomonas aeruginosaClass D carbapenemasesβ-lactam resistanceβ-lactam/β-lactamase inhibitor combination resistancejournal articleopen access10.1128/AAC.01805-25