Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

Abstract

This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays.

Nanoscale battery cathode materials induce DNA damage in bacteria

“…43,44 Since doublestrand breakage is more lethal than single-strand breakage which can be quickly repaired, we used a neutral Comet assay to assess DNA double-strand breakage in bacterial cells, and the tail lengths were used to characterize the extent of damage. 28,45 We treated cells with 5 or 50 mg L À1 nano-NMC at the midlog phase and collected bacterial cells 8 hours (S. oneidensis) or 5 hours (B. subtilis) post-exposure, matching the conditions of toxicity measurements. Fig.…”

“…38 Comet assay for DNA strand breakage Single cell gel electrophoresis on NMC-treated S. oneidensis was conducted following published protocols with minor changes. 45,71 Bacterial cells at mid-log phase were exposed to 5 mg L À1 and 50 mg L À1 NMC for 8 or 5 hours for S. oneidensis or B. subtilis, respectively. Ten microliters of the NMC-treated cell suspension were mixed in 100 mL of 0.5% low-melting agarose (LMA) solution.…”

DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

“…To investigate the possible DNA damage in XTG102, we conducted a comet assay; this method is also referred to as the single-cell gel electrophoresis assay 26 . The alkaline comet assay (pH > 12.3) and the neutral comet assay (pH < 10) are employed to differentiate single-strand and double-strand breaks, respectively 27 . In contrast to wild-type S. enterica cells, XTG102 displayed pronounced DNA double-strand breaks in the neutral comet assay.…”

“…The comet assay was performed essentially as described by Dipesh Solanky et al 39 . Slides were precoated with 0.5% regular agarose and then covered with a 24 mm × 24 mm cover glass.…”

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

“…It can be detected indirectly, through transcriptional fusion of a reporter gene to a promoter that is induced following DNA damage such as alkA or nrdA , and genes belonging to the SOS response ( umuDC, sulA, recN, recA ) (reviewed in (1)). Genotoxicity can also be detected physically through visualization of DNA damage (breaks or rearrangements) (7). In addition to prokaryotic systems, a variety of eukaryotic organisms, notably yeast, Drosophila , and mouse, have also been used as reporters for genotoxocity.…”