Accelerating antimicrobial susceptibility testing (AST) is a priority in the development of novel microbiological methods. The MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) has recently been described as a rapid phenotypic AST method. In this proof-of-principle study, we expanded this method to simultaneously test 24 antimicrobials. An Enterobacterales panel was designed and evaluated using 24 clinical isolates. Either one or two (only for antimicrobials with the EUCAST "I" category) breakpoint concentrations were tested. Microdroplets containing bacterial suspensions with antimicrobials and growth controls were incubated directly on the spots of a disposable MALDI target inside a humidity chamber for 6, 8 or 18 h. Broth microdilution was used as the standard method. After 6 and 8 h of incubation, the testing was valid (i.e., growth control was successfully detected) for all isolates and the overall categorical agreement was 92.0% and 92.7%, respectively. Although the overall assay performance applying short incubation times is promising, the lower performance with some antimicrobials and when using the standard incubation time of 18 h indicates the need for thorough standardization of assay conditions. While using "homebrew" utensils and provisional evaluation algorithms here, technical solutions such as dedicated incubation chambers, tools for broth removal and improved software analyses are needed.
Keywords: Enterobacterales; MALDI-TOF; direct-on-target microdroplet growth assay; multiple antibiotics; multiplex testing; rapid susceptibility testing.