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


RESULTS
Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 microg/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 microg/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 microg/ml but scarce after 10 microg/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.


CONCLUSIONS
This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.
Tamayo
María
M
INIBIC-Complejo Hospitalario Universitario A Coruña, Unidad de Genética, A Coruña, Spain. mtamnov@canalejo.org
Santiso
Rebeca
R
Gosalvez
Jaime
J
Bou
Germán
G
Fernández
José Luis
JL
D000890:Anti-Infective Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004269:DNA, Bacterial / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D008856:Microscopy, Fluorescence; D013997:Time Factors
D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D002939:Ciprofloxacin