Author(s): Walberg M, Gaustad P, Steen HB
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Abstract Exponentially growing E. coli cells were cultivated in the presence of ceftazidime, ciprofloxacin, and gentamicin in concentrations ranging from 0.5-8 minimal inhibitory concentration (MIC), permeabilized by means of cold shock in EDTA/azide, and stained with the DNA-specific dye combination of ethidium bromide and mithramycin before the fluorescence, light scattering, and cell number were measured flow-cytometrically. In order to evaluate the applicability of the cold-shock procedure, cells were also permeabillized by 70\% ethanol. Permeabilization by cold shock, which eliminates washing of the cells, reduced the preparation time to <5 min. A statistically significant increase in 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 concentrations. 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. In gentamicin-treated cells fixed in ethanol, there was no consistent effect on either light scattering or fluorescence; however, we observed a substantial fragmentation and leakage of DNA in such cells. The cell proliferation was completely inhibited within 30 min of gentamicin incubation. For all three drugs, loss of light scattering and DNA were associated with cellular disintegration, i.e., reduced viability. The present results demonstrate that effects of ceftazidime, ciprofloxacin, and gentamicin on E. coli can be detected by flow cytometry within 1 h from the beginning of drug exposure to the completed measurement.
This article was published in Cytometry
and referenced in Journal of Molecular Biomarkers & Diagnosis