Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

Abstract

BackgroundFluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide.ResultsExposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose.ConclusionThis procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

“…24 E. coli DNA histograms corresponding to ciprofloxacin bactericidal concentration showed similar fluorescence intensities throughout the 6 h of incubation. Because of the high ciprofloxacin concentrations used, cellular repair mechanisms were not able to increase DNA synthesis 26 and, therefore, a complete inhibition of DNA synthesis is observed.…”

“…However, membrane potential is reversible, and other studies have shown that depolarized cells are able to grow when placed in fresh medium; 23 thus, the percentage of stained cells will not reflect bacterial viability. It is known that ciprofloxacin has a direct action on chromosomal DNA replication and fragmentation; 24 hence, we intended to evaluate its activity by flow cytometric analysis of intracellular of DNA content using DCR.…”

“…Because of the high ciprofloxacin concentrations used, cellular repair mechanisms were not able to increase DNA synthesis 26 and, therefore, a complete inhibition of DNA synthesis is observed. Furthermore, the lower fluorescence intensities obtained and higher fluorescence broadening without visible peaks could be related to DNA fragmentation, 27 because DNA fragments have a compromised ability to be stained with intercalating dyes, 24 such as DCR, resulting in lower relative DCR fluorescence. These results were further supported by microscopic images, which showed a low staining level and the inability of visualizing chromosomes.…”

Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of Escherichia coli

“…The response to CIP-induced damage is quantified simply by measuring the widths of the diffusion zones of the DNA fragments emerging from the border of the nucleoid. Five different categories of DNA fragmentation were determined, according to the width of the diffusion zone: grade 0, no damage; grade I, low level of damage; grade II, intermediate damage; grade III, high level of damage; and grade IV, massive fragmentation (12).…”

Rapid Determination of Colistin Resistance in Clinical Strains of Acinetobacter baumannii by Use of the Micromax Assay

“…For ciprofloxacin assessment, the lysis procedure removes the cell wall in all bacteria. Those affected by the quinolone, i.e., with doublestrand DNA breaks as a consequence of DNA gyrase and topoisomerase IV trapping, show diffusion of DNA fragments emerging from the residual central core, whereas resistant strains reveal only intact nucleoids (29)(30)(31).…”