title: DNA fragmentation in microorganisms assessed in situ.
abstract: Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.
authors.affiliation: INIBIC-Genética, Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, 15006-A Coruña, Spain. JLFernandez@canalejo.org
authors.forename: José Luis
authors.initials: JL
authors.lastname: Fernández
authors.forename: Mónica
authors.initials: M
authors.lastname: Cartelle
authors.forename: Lourdes
authors.initials: L
authors.lastname: Muriel
authors.forename: Rebeca
authors.initials: R
authors.lastname: Santiso
authors.forename: María
authors.initials: M
authors.lastname: Tamayo
authors.forename: Vicente
authors.initials: V
authors.lastname: Goyanes
authors.forename: Jaime
authors.initials: J
authors.lastname: Gosálvez
authors.forename: Germán
authors.initials: G
authors.lastname: Bou
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000235:genetics*; D004249:DNA Damage / Q000187:drug effects; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D009930:Organic Chemicals; D013194:Staining and Labeling / Q000379:methods; D015003:Yeasts / Q000187:drug effects / Q000235:genetics*
chemical_list: D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D004271:DNA, Fungal; D009930:Organic Chemicals; C474470:SYBR Gold nucleic acid gel stain