Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet...

We aimed to develop a universal phenotypic method, which allows easy and rapid antimicrobial susceptibility testing independently of underlying resistance mechanisms.

We established a novel direct-on-target microdroplet growth assay for the detection of antibiotic resistance within a few hours, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microorganisms were incubated with and without meropenem in nutrient broth as microdroplets directly on MALDI-TOF MS target. Subsequently, broth was separated from microbial cells by contacting the microdroplets with an absorptive material. The microorganisms grown in the presence of antibiotic were detected by MALDI-TOF MS. A total of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa isolates were used to assess performance for detection of meropenem resistance. The microdroplet volumes investigated were 2, 4, 6, 8 and 10 μL.

The best performance was achieved using 6-μL microdroplets. Applying this volume, all growth controls were successfully detected (definition of valid test), and all isolates were correctly categorized as susceptible or non-susceptible after an 18-h incubation. For K. pneumoniae, rate of valid tests, sensitivity and specificity all reached 100% after a 4-h incubation of 6-μL microdroplets. Using the same microdroplet volume for P. aeruginosa, incubation for 5 h resulted in 83.3% of valid tests with 100% sensitivity and 100% specificity.

We demonstrated easy, rapid and accurate resistance detection using carbapenem-resistant Gram-negative bacteria as an example. Our technology is suitable for automatization and expandable to further applications, e.g. simultaneous testing of multiple antibiotics as well as resistance determination directly from clinical samples.