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

Exposing 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.This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.Fluoroquinolones are broad-spectrum antibacterial agents that are used widely to treat a variety of infections, such as gonococcal infections, osteomyelitis, enteric, and respiratory and urinary tract infections. Ciprofloxacin (CIP) is one of the most consumed fluoroquinolones worldwide [1,2]. The type II topoisomerases DNA gyrase and topoisomerase IV are the target of quinolones [3,4]. 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 in