Dry Plate-Based Direct-On-Target Microdroplet Growth Assay for Rapid Antimicrobial Susceptibility Testing of MRSA

Rapid antimicrobial susceptibility testing (AST) is crucial for the early optimization of treatment against infections caused by drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Direct-on-target microdroplet growth assay (DOT-MGA), which uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), has emerged as a promising phenotypic method for rapid AST. The present study aimed to evaluate the feasibility of DOT-MGA for resistant isolates, including the optimi-zation of test conditions.

The feasibility and performance of DOT-MGA were evaluated using a commercial dry plate (DP42) for the AST of MRSA strains, including a clinical isolate with low susceptibility to daptomycin (ATCC 29213, ATCC 43300, and KAM784). DOT-MGA was performed under varying conditions (standard or 10-fold inoculum; 6/8-hour incubation). The minimum inhibitory concentrations (MICs) were compared with those from the reference DP42 method based on their agreement and deviation.

DOT-MGA successfully estimated MICs within 6 - 8 hours. With the standard inoculum, the assay exhibited high concordance with the DP42 results, particularly for the ATCC strains. In contrast, a 10-fold increase in inoculum led to increased MIC values, consistent with the inoculum effect. The daptomycin-low-susceptibility strain (KAM784) exhibited decreased MICs in standard inoculum and short incubation periods, suggesting a risk of under detection in resistant strains. MIC agreement was influenced by both the antibiotic type and MIC rela-tive to the DP42 test range.

DOT-MGA is a rapid phenotypic AST method for MRSA that can be implemented using MALDI-TOF MS and commercial reagents. Optimization of the inoculum size and incubation time is necessary to ensure accuracy, particularly for detecting reduced susceptibility to key antibiotics such as daptomycin and linezolid.