Evaluation of two gradient diffusion tests to determine susceptibility to aztreonam and ceftazidime–avibactam in combination

The combination of aztreonam and ceftazidime-avibactam (ATM-CZA) is a last resort regimen against recalcitrant infections caused by metallo-β-lactamase (MBL)-producing organisms. Susceptibility testing is warranted due to emerging resistance to the combination, but there are no widely implemented methods for use in clinical laboratories. Here, we used a cohort of 100 Enterobacterales<i>, Pseudomonas aeruginosa</i>, and <i>Stenotrophomonas maltophilia</i> strains, including 68 MBL producers, to evaluate the performance of two ETEST strip-based synergy testing methods: the side-by-side (SS) method with an ATM ETEST placed next to a CZA ETEST (10 mm apart) and the strip cross (SX) method with a CZA ETEST placed perpendicularly on top of the ATM ETEST (at the 8 µg/mL mark). By reference broth microdilution (BMD), 89.1% (41/46) of the Enterobacterales, 15% (3/20) of the <i>P. aeruginosa</i>, and 97.1% (33/34) of the <i>S. maltophilia</i> isolates tested susceptible to the ATM-CZA combination. The SS method yielded 72% categorical agreement with BMD and 28 major errors (ME, 36.4%). Initial testing with the SX method yielded three ME , of which one was resolved upon repeat testing, yielding a final categorical agreement of 98% with BMD with two ME (2.6%). The SX method also yielded 100% reproducibility across three brands of Mueller Hinton agar (BD, Hardy, Remel). Our study demonstrates that the SX method is accurate, precise, and feasible for clinical laboratories to perform ATM-CZA susceptibility testing to guide use of the combination for treatment of multidrug-resistant gram-negative pathogens.