Evaluation of Susceptibility Testing Methods for Aztreonam and Ceftazidime-Avibactam Combination Therapy on Extensively Drug-Resistant Gram-Negative Organisms

Carbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa (CR-PA) producing metallo-β-lactamases (MBLs) cause severe nosocomial infections with no defined treatment. The combination of aztreonam (ATM) with ceftazidime-avibactam (CZA) is a potential therapeutic option, but there is no approved, feasible testing method for use in clinical laboratories to assess the activity of two antimicrobials in combination. Here, we evaluate the performance of four ATM-CZA combination testing methods, as follows: broth disk elution (DE), disk stacking (DS), strip stacking (SS), and strip crossing (SX). We used 10 clinical, representative Enterobacterales and 6 P. aeruginosa isolates harboring MBL, Guiana extended-spectrum beta-lactamase (GES), or non-MBL enzymes. Four of these isolates were from clinical cases treated by ATM-CZA. All CRE producing NDM and CR-PA producing GES that were resistant to ATM and CZA alone were susceptible to the ATM-CZA combination. P. aeruginosa generating NDM or VIM remained resistant to ATM-CZA, likely due to non-β-lactamase mechanisms, and all other isolates were susceptible to ATM or CZA alone. The most accurate, precise, and reproducible methods of low complexity were disc elution and both strip methods (SX and SS) using MIC test strips (MTS) , all with 100% sensitivity and specificity, followed by Etest with SX (95.83% sensitivity, 100% specificity) and SS (87.5% sensitivity, 100% specificity). DS had the lowest performance. DE is particularly valuable in low-resource settings that routinely use disks. MTS yielded higher categorical agreements by SX (94%) and SS (84%), relative to Etest by SX (90%) and SS (82%). P. aeruginosa results yielded the majority of the errors. These methods may allow laboratories to inform clinical decision making like combination therapy for severe infections caused by extensively drug-resistant Enterobacterales.