Fast Detection of DNA Damage

Fátima Otero, Rebeca Santiso, María Tamayo, Germán Bou, Jaime Gosálvez, José Luis Fernández

The emergence of multidrug resistant microorganisms together with the decline in discovery and development of new antibiotics is of great concern in health-care policy. In this alarming context, an early and well-tailored antibiotic therapy is a relevant strategy not only to improve clinical outcome but also to avoid appearance and spreading of perilous resistant strains. One of the most common antibiotic classes is fluoroquinolones. They trap the DNA girase and/or topoisomerase IV on the DNA, resulting in DNA fragmentation. We have developed the Micromax(®) assay to determine, in situ, the integrity of the chromosomal DNA-nucleoid from microorganisms. This was validated as a simple procedure for the rapid assessment of the susceptibility or resistance to quinolones in gram-negative bacteria. After incubating with the quinolone, cells are trapped in an agarose microgel on a slide and incubated with a specific lysing solution to remove the cell wall and visualize the nucleoids under fluorescence microscopy. If the strain is susceptible to the quinolone, the bacterial nucleoids show a halo of diffusing DNA spots of fragmented DNA, whereas they appear intact in the resistant strain. The technical processing is performed in 40 min with practically total sensitivity and specificity.