Affiliation:
1. Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
2. Biological Function Research Department, Food Science & Technology Institute, Morinaga Milk Industry Co., Ltd., 5-1-83, Higashihara, Zama, Kanagawa 228-8583, Japan
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Cited by
84 articles.
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