Infections caused by ceftolozane/tazobactam and ceftazidime/avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane/tazobactam and ceftazidime/avibactam resistance mechanisms in all MDR/XDR P. aeruginosa isolates recovered during one year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane/tazobactam and ceftazidime/avibactam resistance were evaluated. Clinical conditions, previous positive cultures and β-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. MLSTs and resistance mechanisms were determined using short- and long-read WGS. The impact of PDCs on β-lactam resistance was demonstrated by cloning into an ampC -deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired β-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane/tazobactam or ceftazidime/avibactam. Seven isolates from different STs owed their β-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219 and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13), and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlight that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

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