Adaptation of Bacterial Comet Assays to Detect Antimicrobial-mediated DNA Strand Breaks in Escherichia coli

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Description
This study was conducted as part of an underlying initiative to elucidate the mechanism of action of natural antibacterial clay minerals for application as therapeutic agents for difficult-to-treat infections such as methicillin-resistant Staphylococcus aureus (MRSA)-derived skin lesions and Buruli ulcer.

This study was conducted as part of an underlying initiative to elucidate the mechanism of action of natural antibacterial clay minerals for application as therapeutic agents for difficult-to-treat infections such as methicillin-resistant Staphylococcus aureus (MRSA)-derived skin lesions and Buruli ulcer. The goal of this investigation was to determine whether exposure to the leachate of an antibacterial clay mineral, designated as CB, produced DNA double-strand breaks (DSBs) in Escherichia coli. A neutral comet assay for bacterial cells was adapted to assess DSB levels upon exposure to soluble antimicrobial compounds. Challenges involved with the adaptation process included comet visualization and data collection. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions comprised of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to CB resulted in significantly longer comet lengths compared to negative control exposures, suggesting that CB killing activity involves the induction of DNA DSBs. The results of this investigation further characterize the antimicrobial mechanisms associated with a particular clay mineral mixture. The adapted comet assay protocol described herein functions as an effective tool to assess double-strand fragmentation resulting from exposure to soluble antimicrobial compounds and to visually compare results from experimental and control reactions.
Date Created
2012-12
Agent

Metal Ions, Not Metal-Catalyzed Oxidative Stress, Cause Clay Leachate Antibacterial Activity

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Description

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions.

Date Created
2014-12-11
Agent