Rapid Direct Susceptibility Testing from Positive Blood Cultures by the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay

Abstract

The recently developed direct-on-target microdroplet growth assay (DOT-MGA) allows rapid universal antimicrobial susceptibility testing (AST) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Here, we investigated a direct application of this method on positive blood cultures (BCs) for the acceleration of sepsis diagnostics. Blood samples spiked with meropenem-nonsusceptible and meropenem-susceptible Enterobacterales isolates were inoculated into Bactec Plus Aerobic/F bottles and incubated in the Bactec automated system. Positive-BC broth was processed using four different methods, filtration/dilution, dilution, lysis/centrifugation, and differential centrifugation. For both dilution-based methods, AST was performed from 1:100, 1:1,000, and 1:10,000 dilutions of positive-BC broth in cation-adjusted Mueller-Hinton broth (CA-MHB). For both centrifugation-based methods, a 0.5 McFarland standard turbidity suspension was prepared from a bacterial pellet and adjusted to a final inoculum of 5 × 10⁵ CFU/ml in CA-MHB. Six-microliter microdroplets with or without meropenem at the breakpoint concentration were spotted in triplicate onto a MALDI-TOF MS target, followed by incubation in a humidity chamber for 3 or 4 h and subsequent broth removal. Spectra were evaluated by MALDI Biotyper software. The test was considered valid if the growth control without antibiotic achieved an identification score of ≥1.7. For samples with meropenem, successful identification (score, ≥1.7) was interpreted as a nonsusceptible result, whereas failed identification (score, <1.7) defined susceptibility. The best test performance was achieved with the lysis/centrifugation method after a 4-h incubation. At this time point, 96.3% validity, 91.7% sensitivity, and 100% specificity were reached. This study demonstrated the feasibility and accuracy of a rapid DOT-MGA from positive BCs. Parallel to susceptibility determination, this method provides simultaneous species identification.

Keywords: AST; MALDI-TOF; blood culture; carbapenem resistance; direct-on-target microdroplet growth assay; rapid susceptibility testing.