Abstract
Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-β-lactamase blaVIM-20 and the narrow-spectrum oxacillinase blaOXA-2 were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the blaOXA-2 β-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin-β-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa.