TY - JOUR
T1 - Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli
AU - Solanky, Dipesh
AU - Haydel, Shelley
N1 - Funding Information:
We would like to thank Caitlin Otto and Andrea Loes for their assistance in the experimental design process and for providing helpful discussions and critical evaluation of the manuscript. This research was supported by Public Health Service grant AT004690 (awarded to S.E.H.) from the NIH National Center for Complementary and Alternative Medicine .
PY - 2012/11
Y1 - 2012/11
N2 - This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated as 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 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.
AB - This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated as 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 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.
KW - Antibacterial mineral leachate
KW - Antibiotic
KW - DNA double-strand breaks
KW - E. coli
KW - Neutral comet assay
KW - Single cell gel electrophoresis
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U2 - 10.1016/j.mimet.2012.08.009
DO - 10.1016/j.mimet.2012.08.009
M3 - Article
C2 - 22940101
AN - SCOPUS:84867798579
SN - 0167-7012
VL - 91
SP - 257
EP - 261
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 2
ER -