17 A rapid, reproducible, high-throughput inkjet checkerboard antimicrobial susceptibility detection method to identify effective combination therapies for M. abscessus infection

Background Mycobacterium abscessus lung and skin infections are highly morbid and notoriously difficult to treat, with cure rates for pulmonary infections ranging from only 30-50%. Treatment regimens typically involve three or more drugs administered for months, often resulting in significant toxicity. Antimicrobial susceptibility testing (AST) and synergy testing have the potential to enhance patient outcomes by guiding more effective treatment strategies. However, conventional checkerboard assays, commonly used for synergy testing, are highly labor-intensive, limiting research progress. Additionally, testing individual or combination regimens for patient isolates requires long turnaround times and is highly operator-dependent. As a result, synergy testing remains beyond the capability of most clinical and research laboratories and is not readily available to inform direct patient care. This study aims to develop a rapid, high-throughput checkerboard synergy detection method to make data-driven synergy testing more accessible and reproducible. Methods Checkerboard synergy testing was performed on pairwise and triple-drug combinations, including omadacycline, azithromycin, and clofazimine; omadacycline, clarithromycin, and clofazimine; and bedaquiline, imipenem, and linezolid against two M. abscessus clinical isolates. Resazurin dye was added to facilitate visual readout. A digital dispenser inkjet printing technology was used to establish and evaluate simplified diagonal fractional inhibitory concentration (FIC) grids. Results The checkerboard assay with resazurin provided visually interpretable results by Day 3, which were consistent with the standard Day 5 results obtained through CLSI procedures without resazurin. Strong correlations were observed between the plate reader readout on Day 3 and the Day 5 CLSI results. Multiple synergistic drug combinations were identified, including omadacycline, azithromycin, and clofazimine; omadacycline, clarithromycin, and clofazimine; as well as bedaquiline and linezolid. Conclusions The addition of resazurin enables a faster readout, allowing for the detection of synergy results two days earlier than conventional methods. This approach offers a more efficient AST and checkerboard synergy testing strategy, potentially improving the speed and accessibility of synergy testing for M. abscessus infections.