Laser-induced fluorescence - Capillary electrophoresis and fluorescence microplate reader measurement: Two methods to quantify the effect of antibiotics

Putative antibiotic drugs have to be classified according to their bactericidal potential. Two new methods by means of fluorescence spectroscopy (FS) using a fluorescence microplate reader (FMR) and laser-induced fluorescence capillary electrophoresis (LIF-CE), respectively, were developed for the assessment of the bactericidal efficiency using the LIVE/DEAD BacLight viability kit composed of the two fluorescent nucleic acid stains, SYTO9 (stains all cells green) and propidium iodide (stains cells with damaged membrane red). By correlation of the ratio of green and red fluorescence with the percentage of live cells by employing FS and LIF-CE, respectively, dose response curves of drug-treated Pseudomonas spp. and Streptococcus spp. samples were calculated, which allowed for the assessment of ED50 values. Both methods led to similar results which were in agreement with the minimum inhibitory concentrations (MICs) obtained by conventional broth microdilution. The application of the BacLight viability kit on drug-treated bacteria cultures presents a rapid method of assessing the antibiotic potency which is of great importance for high throughput screening in the development of new antibiotics. Additionally, the new LIF-CE method, which based on the use of a second unlabeled bacteria injection as a stacking front, allowed drawing conclusions from the electrophoretic profile about the constitution of the bacterial population. Thus, the tendency of bacterial chain formation and alterations in the live/dead ratio of the bacterial composition can be directly observed in the presence of different antibiotics.