Flow Cytometric Bacterial Susceptibility Testing

摘要

Abstract Exponentially growing E. coli cells were cultivated in the presence ofceftazidime, ciprofloxacin, and gentamicin in concentrations ranging from 1/2 to 8 MIC, permeabilized by means of cold shock in EDTA/Na-azide, stained with the DNA specific dye combination of ethidium bromide and mithramycin before the fluorescence, light scattering, and cell number was measured flow cytometrically. In order to evaluate the applicability of the cold shock procedure, cells were also permeabilized by 70 % ethanol. Permeabilization by cold shock, which eliminates washing of the cells, reduced the preparation time to less than 5 min. A statistically significant increase of the light scattering and fluorescence, i.e. cell size and DNA content, could be detected already after 30 min of ceftazidime and ciprofloxacin exposure, even at sub-MiC concentration. The results obtained with these drugs with cold shock permeabilization were similar to those seen with ethanol fixation. For gentamicin treated cells, however, a majority of the cells lost their fluorescence after cold shock, indicating substantial fragmentation and leakage of DNA by this drug. In gentamicin treated cells fixed in ethanol there was no consistent effect on either light scattering or fluorescence, while cell proliferation was completely inhibited within 30 min of incubation. The present results demonstrate that effects of ceftazidime, ciprofloxacin, and gentamicin on E. coli can be detected by flow cytometry within one hour from the beginning of drug exposure to the finished measurement, and with a sensitivity on par with or better than that of conventional plaque assays, which typically require 24 hours or more.