Phenotypic screening for metallo-β-lactamases and extended-spectrum β-lactamases in Pseudomonas aeruginosa using the VITEK-2 AST-N443 and Phoenix NMIC-486 cards.

Pseudomonas aeruginosa can develop carbapenem resistance through chromosomal mechanisms and/or by acquiring plasmid-mediated carbapenemases, including the most prevalent metallo-β-lactamases (MBLs) and/or extended-spectrum β-lactamases (ESBLs). Conventional β-lactam susceptibility testing does not reliably discriminate MBL/ESBL-producing carbapenem-resistant P. aeruginosa (MBL/ESBL-CRPA) from non-carbapenemase-producing isolates, here referred to as AmpC-hyperproducers. However, these groups exhibit distinct susceptibility patterns to novel β-lactam/β-lactamase inhibitor (BL/BLI) combinations, including ceftolozane/tazobactam (C/T), ceftazidime/avibactam (C/A), imipenem/relebactam (I/R), and meropenem/vaborbactam (M/V). We investigated potential screening cut-offs for detecting MBL/ESBL production in CRPA using the VITEK-2 AST-N443 and Phoenix NMIC-486 cards. Antimicrobial susceptibility testing was performed using the VITEK-2 AST-N443 and Phoenix NMIC-486 cards on 135 whole-genome-sequenced international CRPA isolates resistant by disc diffusion to ceftazidime, cefepime, imipenem, and/or meropenem. The collection included 71 MBL-producing isolates, of which nine co-produced ESBLs, as well as one KPC-2-, one GES-5-, and twelve ESBL-only producers. The remaining 50 represented AmpC-hyperproducers. MIC distributions were analyzed to identify discriminatory screening cut-offs. C/T was the most discriminatory BL/BLI combination for differentiating MBL/ESBL-CRPA from AmpC-hyperproducers. For the VITEK-2 AST-N443, a C/T MIC ≥32 mg/L achieved 100% sensitivity for MBL±ESBL detection (71/71) and 91.7% sensitivity for ESBL-only producers (11/12), with 82% specificity (41/50). For the Phoenix NMIC-486, a C/T MIC >8 mg/L achieved 100% sensitivity for both MBL and ESBL detection, with 80% specificity (40/50). Combining additional BL/BLI agents did not further improve specificity. Elevated C/T MICs represent reliable phenotypic screening markers for MBL and ESBL production in CRPA using both the VITEK-2 AST-N443 and Phoenix NMIC-486 systems.IMPORTANCERapid identification of the underlying resistance mechanism in carbapenem-resistant Pseudomonas aeruginosa (CRPA) is essential for effective treatment and infection control. Differentiating high-level resistance acquired via plasmid-borne carbapenemases and/or extended-spectrum β-lactamases (ESBLs) from resistance driven by intrinsic mechanisms, including AmpC-type β-lactamase hyperproduction, remains challenging because conventional antimicrobial susceptibility testing (AST) using classic β-lactams cannot reliably separate these phenotypes. This distinction is relevant because many recently introduced β-lactam/β-lactamase inhibitor combinations are inactive against metallo-β-lactamase (MBL) carbapenemase-producing isolates but may remain active against strains in which resistance is driven by intrinsic mechanisms. Detection of acquired carbapenemases may also trigger enhanced infection-control measures and epidemiological investigations. Our study identifies reliable screening cut-offs for MBL- and ESBL-production in CRPA using standard VITEK 2 and Phoenix automated AST panels. This readily available approach may accelerate detection, support routine laboratory workflows, reduce unnecessary confirmatory testing, and facilitate earlier targeted therapy and infection-control interventions in laboratories using these systems worldwide without requiring additional specialized testing or infrastructure.