Soejima Takashi, Iida Ken-ichiro, Qin Tian, Taniai Hiroaki, Seki Masanori, Yoshida Shin-ichi
Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
J Clin Microbiol. 2008 Jul;46(7):2305-13. doi: 10.1128/JCM.02171-07. Epub 2008 Apr 30.
Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 10(8) heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 10(8) heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 10(3) to 10(7) antibiotic-treated L. monocytogenes cells but could detect 10(2) live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 10(3) to 10(7) heat-treated cells but could detect 10(1) live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.
单叠氮溴化乙锭（EMA）是一种DNA交联剂和真核拓扑异构酶II毒药。我们之前报道过，用可见光照射EMA（EMA + 光）可直接切割大肠杆菌的染色体DNA（T. Soejima、K. Iida、T. Qin、H. Taniai、M. Seki、A. Takade和S. Yoshida，《微生物学与免疫学》51:763 - 775，2007年）。在此，我们报告EMA + 光在处理10分钟内可随机切割经热处理但非活的单核细胞增生李斯特菌细胞的染色体DNA。当对大小为894 bp的DNA进行PCR扩增时，来自10⁸个经热处理的单核细胞增生李斯特菌的PCR终产物被EMA + 光完全抑制。当靶DNA较短（113 bp），如单核细胞增生李斯特菌的hly基因时，仅EMA + 光并不能完全抑制DNA扩增。因此，我们将DNA促旋酶/拓扑异构酶IV和哺乳动物拓扑异构酶毒药（此处简称为T - 毒药）与EMA + 光一起使用。T - 毒药可在30分钟内穿透经热处理但非活的单核细胞增生李斯特菌细胞，通过中毒活性切割染色体DNA。来自10⁸个经热处理的单核细胞增生李斯特菌细胞的hly基因的PCR产物受到EMA + 光和T - 毒药组合（EMA + 光 + T - 毒药）的抑制，但来自活细菌的PCR产物未被抑制。作为临床应用于菌血症的模型，我们试图区分人血液中存在的活的和经抗生素处理的单核细胞增生李斯特菌细胞。EMA + 光 + T - 毒药完全抑制了来自10³至10⁷个经抗生素处理的单核细胞增生李斯特菌细胞的PCR产物，但能检测到10²个活细菌。考虑到食物中毒的预防和控制，该方法被应用于区分添加到巴氏杀菌牛奶中的活的和经热处理的单核细胞增生李斯特菌细胞。EMA + 光 + T - 毒药抑制了来自10³至10⁷个经热处理细胞的PCR产物，但能检测到10¹个活的单核细胞增生李斯特菌细胞。我们的方法在临床以及食品卫生检测中都很有用。
