Rapid Simultaneous Testing of Multiple Antibiotics by the MALDI-TOF MS Direct-on-Target Microdroplet Growth Assay

Area of Science:

• Microbiology
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Accelerating antimicrobial susceptibility testing (AST) is crucial for effective treatment of bacterial infections.
• Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) offers rapid bacterial identification.
• The direct-on-target microdroplet growth assay (DOT-MGA) is a novel phenotypic AST method utilizing MALDI-TOF MS.

Purpose of the Study:

• To expand the MALDI-TOF MS DOT-MGA for simultaneous testing of 24 antimicrobials against Enterobacterales.
• To evaluate the performance of this expanded DOT-MGA method using clinical isolates.
• To assess the impact of incubation time on assay accuracy.

Main Methods:

• Development and evaluation of a 24-antimicrobial panel for DOT-MGA.
• Testing of 24 clinical Enterobacterales isolates with one or two breakpoint concentrations.
• Incubation of microdroplets on MALDI target plates within a humidity chamber for 6, 8, or 18 hours.
• Comparison with broth microdilution as the standard method.

Main Results:

• The DOT-MGA assay demonstrated high overall categorical agreement: 92.0% at 6 hours and 92.7% at 8 hours.
• Assay validity was confirmed for all isolates at shorter incubation times (6 and 8 hours).
• Lower performance was observed with certain antimicrobials and at the standard 18-hour incubation time.

Conclusions:

• The expanded MALDI-TOF MS DOT-MGA is a promising rapid phenotypic AST method for Enterobacterales.
• Short incubation times (6-8 hours) yield promising results, but standardization is needed for consistent performance.
• Further technical development, including dedicated equipment and improved algorithms, is required for clinical implementation.