Functional characterization of a novel class A carbapenemase CAE-1 in carbapenem-resistant <i>Pseudomonas aeruginosa</i> clinical isolates

Two carbapenem-resistant <i>Pseudomonas aeruginosa</i> (CRPA) isolates were collected, which lacked known carbapenemases but produced a β-lactamase CAE-1. CAE-1 was recently found in <i>Comamonas aquatica</i> and conferred resistance to penicillins and cephalosporins. In this study, we aim to know whether CAE-1 was responsible for the carbapenem resistance in the CRPA isolates and its enzyme hydrolyzing characteristics. Both CRPA clinical isolates exhibited minimal inhibitory concentrations (MICs) of 8 µg/mL for imipenem and meropenem, with a positive result in the modified Hodge test for meropenem. The <i>bla</i>_CAE-1 and <i>bla</i>_KPC-2 were cloned and expressed in <i>P. aeruginosa</i> PAO1 and <i>Escherichia coli</i> DH5α, respectively. The <i>bla</i>_CAE-1-carrying PAO1 transformant also tested positive using the modified carbapenem inactivation method and was resistant to piperacillin-tazobactam, ticarcillin-clavulanate, and cefoperazone-sulbactam, but susceptible to ceftazidime-avibactam. Expression of <i>bla</i>_CAE-1 in <i>P. aeruginosa</i> PAO1 elicited a 64- to 128-fold increase in MICs for piperacillin, ceftazidime, cefepime, and aztreonam, and an eightfold increase for meropenem, exhibiting a broader resistance profile than in <i>E. coli</i> DH5α. Steady-state kinetic assays showed that CAE-1 had catalytic efficiency against all β-lactams tested, with comparatively lower efficiency against three carbapenems relative to KPC-2, while demonstrating approximately equivalent efficiency for the other β-lactam antibiotics tested. Whole-genome sequencing revealed that <i>bla</i>_CAE-1 was a class A β-lactamase and encoded on an integrative and conjugative element, which might facilitate its horizontal transfer. The class A β-lactamase CAE-1 is a carbapenemase posing a high risk for horizontal dissemination. Enhanced surveillance for <i>bla</i>_CAE-1-harboring isolates is needed.