In vivo emergence of ceftazidime/avibactam resistance in Pseudomonas aeruginosa driven by OXA-1381, a novel W157L Ω-loop variant of OXA-2

The emergence of ceftazidime/avibactam resistance in an extended-spectrum β-lactamase (ESBL)-producing Pseudomonas aeruginosa isolate during ceftazidime/avibactam therapy was investigated and the underlying mechanisms elucidated. Two sequential isolates recovered before and after the ceftazidime/avibactam treatment were characterized by antimicrobial susceptibility testing, whole-genome sequencing, and functional and biochemical analyses. The initial isolate was susceptible to ceftazidime/avibactam, whereas the later isolate developed resistance (increase in MIC from 2 to 32 mg/L), accompanied by collateral carbapenem susceptibility. Both isolates co-produced a novel BEL variant (designated BEL-5) and an OXA-2 enzyme, encoded by adjacent genes within a class one integron embedded in a 31,623-bp IncP plasmid. The resistant isolate harbored a previously undescribed OXA-2 variant, designated OXA-1381, carrying a W157L substitution in the Ω-loop. Cloning experiments demonstrated that OXA-1381 is an extended-spectrum variant conferring increased resistance to ceftazidime/avibactam and ceftolozane/tazobactam but decreased resistance to carbapenems. Kinetic analyses revealed a marked increase in the affinity and catalytic efficiency of OXA-1381 against ceftazidime and ceftolozane, while showing reduced hydrolytic activity against meropenem, consistent with the observed phenotypic trade-off. Inhibition assays showed similar susceptibility to avibactam for both enzymes, confirming that resistance was driven by enhanced hydrolysis rather than by impaired inhibition. The findings document the in vivo evolution of ceftazidime/avibactam resistance in an ESBL-producing P. aeruginosa background, mediated by structural modification of a narrow-spectrum OXA-2 enzyme. In addition, they highlight the potential of integron-borne genes encoding “silent” β-lactamases to evolve under antibiotic pressure and compromise the activity of last-line agents.