Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli.
Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli.
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DOI：
10.1016/j.mimet.2012.08.009
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发表时间：
2012-11
影响因子：
2.2
通讯作者：
Haydel, Shelley E.
中科院分区：
文献类型：
--
作者：
Solanky, Dipesh;Haydel, Shelley E.
关键词：
本研究旨在确定一种天然抗菌粘土矿物混合物，指定CB的作用机制，通过调查在大肠杆菌中的DNA双链断裂（DSB）的诱导。为了量化暴露于可溶性抗微生物化合物后的DNA损伤，我们修改了细菌中性彗星试验，该试验主要将染色染色体或彗星的一般长度与DSB相关DNA损伤的程度相关联。为了适当解释抗菌剂介导的链断裂，开发了适当的对照反应，包括暴露于水、乙醇、卡那霉素和博来霉素，并对试验进行了优化。与水和卡那霉素暴露相比，细菌暴露于CB粘土导致显著更长的彗星长度，这表明DNA DSB的诱导有助于这种抗菌粘土矿物混合物的杀伤活性。本文所述的彗星测定方案提供了用于评价可溶性抗微生物剂衍生的DNA损伤和用于比较实验测定和对照测定之间的DNA片段化的一般技术。
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.
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