Rapidly quantitative antimicrobial susceptibility testing for Klebsiella pneumoniae using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry under optimal conditions

Abstract

Background Klebsiella pneumoniae infections, especially Carbapenem-resistant Klebsiella pneumoniae (CRKP), have become an “Urgent Threats” with high morbidity and mortality. Therefore, rapidly determining of the susceptibility and timely choosing an appropriate antibiotic were the important premises of the treatment of Klebsiella pneumoniae infections. The present study was first to explore the fitness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in quantitative rapid antimicrobial susceptibility testing (RAST) with optimal conditions. Methods Firstly, we optimized the methodology based on the formic acid extraction method. Taking broth micro-dilution method (BMD) as a reference method, we utilized 25-fold, 50-fold and 100-fold dilutions matching with appropriate incubation time to evaluate the consistency of minimum inhibition concentration (MIC) values derived from BMD and MALDI-TOF MS. Finally, the performances of optimal dilution and incubation time were verified through the reproducibility on different three days. Results Directly incubated in the EP tubes and skipped the washing step can not only simplify the procedure, but reduce the unnecessary loss of bacteria biomass to improve sensitivity. The optimal volumes of 70% formic acid/100% acetonitrile (FA/ACN) and supernatant on the target plate were 3 µL and 2 µL, respectively. Comparing the different combination, 25-fold dilution of the 0.5 McFarland suspension and 2hr incubation time resulted in best performance. The consistency between the MS-MIC and corresponding BMD-MIC values in CRKP and Carbapenem-susceptible Klebsiella pneumoniae (CSKP) strains accounted for 76.67% and 66.67%, respectively, which generated the substantial agreements between MS-MIC and BMD-MIC values for CRKP strains (Kappa=0.643) and the moderate agreements for CSKP strains (Kappa=0.476). Finally, the MALDI-TOF MS-based RAST showed a well repeatability. Conclusion Considerable potentials were demonstrated for universal and mechanism-indepent RAST by MALDI-TOF MS with optimal conditions, which strengthened its application in accelerating reporting time and clinical diagnosis.