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Applied and Environmental Microbiology, October 2008, p. 5925-5933, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00318-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

DNA Fragmentation in Microorganisms Assessed In Situ{triangledown}

José Luis Fernández,1,2* Mónica Cartelle,1 Lourdes Muriel,1 Rebeca Santiso,1,2 María Tamayo,1,2 Vicente Goyanes,1 Jaime Gosálvez,3 and Germán Bou4

INIBIC-Genética, Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, 15006-A Coruña, Spain,1 Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, Avda. de Montserrat s/n, 15009-A Coruña, Spain,2 Unidad de Genética, Facultad de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain,3 INIBIC-Microbiología, Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, 15006-A Coruña, Spain4

Received 6 February 2008/ Accepted 3 August 2008

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.

* Corresponding author. Mailing address: Sección de Genética y Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, 15006-A Coruña, Spain. Phone: 34 981 287499. Fax: 34 981 287122. E-mail: JLFernandez{at}canalejo.org

{triangledown} Published ahead of print on 8 August 2008.

Applied and Environmental Microbiology, October 2008, p. 5925-5933, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00318-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Santiso, R., Tamayo, M., Fernandez, J. L., del Carmen Fernandez, M., Molina, F., Villanueva, R., Gosalvez, J., Bou, G. (2009). Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of Escherichia coli. J. Clin. Microbiol. 47: 2593-2595 [Abstract] [Full Text]  


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