RAPID ASSESSMENT OF CEFTAZIDIME, CIPROFLOXACIN, AND GENTAMICIN SUSCEPTIBILITY IN EXPONENTIALLY-GROWING ESCHERICHIA-COLI-CELLS BY MEANS OF FLOW-CYTOMETRY

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 (MIG), permeabilized by me ans of cold shock in EDTA/Na-azide, and stained with the DNA-specific dye combination of ethidium bromide and mithramycin before the fluores cence, Light scattering, and cell number were measured flow-cytometric ally. In order to evaluate the applicability of the cold-shock procedu re, cells were also permeabilized by 70% ethanol. Permeabilization by cold shock, which eliminates washing of the cells, reduced the prepara tion time to <5 min. A statistically significant increase in light sca ttering and fluorescence, i.e., cell size and DNA content, could be de tected already after 30 min of ceftazidime and ciprofloxacin exposure, even at sub-MIG concentrations. The results obtained with these drugs with cold-shock permeabilization were similar to those seen with etha nol 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 ligh t scattering or fluorescence; however, we observed a substantial fragm entation and leakage of DNA in such cells. The cell proliferation was completely inhibited within 30 min of gentamicin incubation. For all t hree drugs, loss of light scattering and DNA were associated with cell ular disintegration, i.e., reduced viability. The present results demo nstrate that effects of ceftazidime, ciprofloxacin, and gentamicin on E. coli can be detected by now cytometry within 1 h from the beginning of drug exposure to the completed measurement. (C) 1997 Wiley-Liss, I nc.