Browsing by Author "Sundberg, Walter"

Time-kill assays are commonly used for determining an antibiotic’s activity against a bacterial strain over time. The assay is carried out by adding an antibiotic to media containing bacteria, and the colony-forming units/ml is determined at different timepoints using the plate count method. Time-kill assays are commonly used for determining synergism between two or more antibiotics, and for determining if an antibiotic has a time- or concentration dependent effect. The aim of this study was to validate a modified time-kill assay. The modified assay is high-throughput screening-compatible, and less reagent- and labour-intensive compared to the traditional method. In this modified assay, the number of viable bacteria is evaluated with resazurin reduction. Metabolically active bacteria reduce resazurin (non-fluorescent) to resorufin (fluorescent) during growth. Resazurin to resorufin reduction occurs faster with increasing amounts of bacteria. The resazurin reduction was measured by two alternative methods: with a spectrophotometer measuring fluorescence and with OmniLog®, an imaging incubator that measures light transmission through the wells of microplates. First, the applicability of the assay to different bacterial species was tested by comparing optical density- and fluorescence-based readings. Then, cold tolerance and plate uniformity assays were carried out, and the minimum inhibitory concentrations of the selected antibiotics were then determined. The traditional method and the resazurin-reduction assay were then performed in parallel. The results showed that the spectrophotometric and OmniLog® measurements worked similarly for Enterococcus faecalis and Klebsiella pneumoniae. The cold treatment did not affect the viability of neither E. faecalis nor K. pneumoniae. With K. pneumoniae, the plates were uniform at both high and low bacterial concentrations, whereas the variability increased at lower bacterial concentrations with E. faecalis. The colony counting assay and the resazurin reduction assay correlated well for five out of the seven antibiotics against E. faecalis. These include the cell wall-active antibiotics ampicillin and vancomycin. When the translation inhibitors tetracycline and chloramphenicol were used, the results obtained from the two methods differed markedly. In conclusion, the correlation between the two methods was dependent on the antibiotics’ mechanism of action.