Takashi Tsugukuni

Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride

We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy.

Method for Rapid Detection and Identification of Chaetomium and Evaluation of Resistance to Peracetic Acid

In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the β-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

Biodegradation and toxicity to fish of di-long-chain tertiary amine salt containing ester and amide bonds

Biodegradability of N-(3-alkanoylaminopropyl)-N-(2-alkanoyloxyethyl)-N- methylammonium chloride (EAA) was investigated. Biodegradabilities by biochemical oxygen demand (BOD) and dissolved organic carbon (DOC) after 28 days were 79 and 91%, respectively, and almost the same amount of ammonium ion as the theoretical value was detected using a modified MITI test (I) (OECD guidelines, 301C). In the test with activated sludge obtained from a municipal sewage treatment plant, biodegradabilities by BOD and DOC after 35 days were 87 and 98%, respectively, and the 1H-NMR analysis of the tested solution which was done separately under similar conditions indicated the rise and fall of two biodegradation intermediates. Therefore, EEA was considered to be a readily and ultimately biodegradable compound. Besides, the 96-hr LC50 value in red killifish (Oryzias latipes) of EAA was 66 mg/liter. More than 1000 mg/liter was of biodegradation intermediates rapidly made by biodegradation of EAA. These results reveal that EAA has sufficient environmental compatibility.