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.
Highlights
Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA
DNA gyrase is the preferential target in gram-negative bacteria such as E. coli, whereas topoisomerase IV is affected mainly in gram-positive bacteria [5]
Cultures Chromosomal DNA fragmentation in situ was assayed in the TG1 E. coli strain, which was grown routinely in Luria Bertani (LB) broth (1% Bacto-tryptone, 0.5% yeast extract, 0.5% NaCl) or on LB agar at 37°C in aerobic conditions
Summary
Fluoroquinolones 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. DNA gyrase is the preferential target in gram-negative bacteria such as E. coli, whereas topoisomerase IV is affected mainly in gram-positive bacteria [5] These enzymes induce transient DNA double-strand breaks (DSBs) on bacterial chromosomes, which either introduce negative supercoiling, as in the case of DNA gyrase, or relax supercoiling and decatenate-replicated daughter chromosomes, as in the case of topoisomerase IV [3,4,5]. A transient covalent link is established between the GyrA or the ParC subunits and the 5' end of each DNA break [3,5]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call