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TDK conference 2023Ecsedi Bence Gergő - year 4 University of Veterinary Medicine Budapest, Department of Pharmacology and Toxicology Supervisor: Dr. Ádám Kerek One of the most important problems of our time is the spread of antimicrobial resistance, for which we test the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values with incubation times of 1 and 3 days. Evolution and co-selection assays, on the other hand, generally require 10–12-day assays. Very little information is available in the literature on the stability of antibiotics in culture media over longer periods. The objective of our study was to determine the stability of eight different antibiotic stock solutions (amoxicillin, cefotaxime, neomycin, oxytetracycline, florfenicol, enrofloxacin, colistin, potentiated sulphonamide) and their 10-base dilution series in culture medium at 37 °C with an incubation time of 12 days. In our study, 40 ml of stock solution of each active substance was prepared, and the 10-base dilution series was then prepared in tryptone-soy broth. Stock solutions and diluted samples were incubated at 37 °C and sampled immediately after preparation and on days 1, 2, 5, 7, 9 and 12. Samples taken on the measurement days were diluted to ensure the presence of the active substances at the appropriate concentration for the measurement using a SCIEX Exion LC™ 2.0 ultra-high-performance liquid chromatography (UHPLC) system coupled to a SCIEX QTRAP 4500 triple quadrupole mass spectrometry system. Of the aqueous stock solutions, neomycin, florfenicol and potentiated sulphonamide solutions were stable (>95%) throughout the study. Florfenicol maintained its stability in the culture medium throughout (100%), potentiated sulphonamide showed a slight degradation (>85%), whereas neomycin showed a significant degradation already on the first day. For amoxicillin, oxytetracycline and colistin, aqueous solutions were more stable than solutions diluted in tryptone-soy broth; however, both aqueous solutions and solutions mixed in the broth showed significant degradation, with an average of 2-25% of the initial concentrations detected by day 12 of the study. Of these, oxytetracycline showed the greatest degradation, with only 2% of the total detectable on average at day 12. Stability was similar for cefotaxime and enrofloxacin in aqueous solutions and in culture medium, with 3.6% of the former and 88.7% of the latter detectable by day 12. Our results are particularly relevant for those active substances that showed significant degradation already after 24 h and are important to consider in MIC studies, even more so in MBC studies; and, in long-term evolution and co-selection. In conclusion, stability studies in culture media are also essential to validate the results in the future. List of lectures |