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Compared to MICs (more than 800 g/ml) of (؊)-epigallocatechin gallate (EGCg) against Escherchia coli, MICs of EGCg against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) were 100 g/ml or less. Furthermore, less than 25 g EGCg per ml obviously reversed the high level resistance of MRSA to all types of tested ␤-lactams, including benzylpenicillin, oxacillin, methicillin, ampicillin, and cephalexin. EGCg also induced a supersusceptibility to ␤-lactams in MSSA which does not express mecA, encoding penicillin-binding protein 2 (PBP2). The fractional inhibitory concentration (FIC) indices of the tested ␤-lactams against 25 isolates of MRSA were from 0.126 to 0.625 in combination with 6.25, 12.5 or 25 g of EGCg per ml. However, no synergism was observed between EGCg and ampicillin against E. coli. EGCg largely reduced the tolerance of MRSA and MSSA to high ionic strength and low osmotic pressure in their external atmosphere, indicating damage of the cell wall. Unlike dextran and lipopolysaccharide, peptidoglycan from S. aureus blocked both the antibacterial activity of EGCg and the synergism between EGCg and oxacillin, suggesting a direct binding of EGCg with peptidoglycan on the cell wall. EGCg showed a synergistic effect with DL-cycloserine (an inhibitor of cell wall synthesis unrelated to PBP2) but additive or indifferent effect with inhibitors of protein and nuclear acid synthesis. EGCg did not suppress either PBP2 mRNA expression or PBP2 production, as confirmed by reverse transcription-PCR and a semiquantitative PBP2 latex agglutination assay, indicating an irrelevance between the synergy and PBP2 production. In summary, both EGCg and ␤-lactams directly or indirectly attack the same site, peptidoglycan on the cell wall. EGCg synergizes the activity of ␤-lactams against MRSA owing to interference with the integrity of the cell wall through direct binding to peptidoglycan.

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Recently green tea, which is the traditional drink of Japan and China, has been recognized as healthful. Catechins, which are polyphenol chemical compounds found in abundance in green tea, possess physiological effects including antioxidative and bactericidal action as well as antitumor activity. Four main catechins are contained within green tea: epicatechin (EC), epicatechin gallate (ECg), epigallocatechin (EGC) and epigallocatechin gallate (EGCg). These catechins display strong antioxidant activity, 1) which efficiently eliminates reactive oxygen species. The antioxidant mechanism is believed to involve radical elimination by the phenolic hydroxyl group of the catechin structure.2) EGCg and EGC in catechin have demonstrated anti-tumor growth and a strong apoptosis induction effect in the human lung tumor cells H661 and H1299 and the induction was inhibited by catalase.3) This paper indicates that hydrogen peroxide participated in the apoptotic action of EGCg; however, the chemical mechanism has not been clarified.On the other hand, we noted previously that catechins exhibit a wide antibacterial spectrum of gram-negative bacteria and positive fungus in terms of germicidal action.4) The structure-activity relationship of catechin and bactericidal activity occurs in the following order: ECgϾEGCgϾEGCϾ EC.5) Furthermore, the gallate and pyrogallol moieties are necessary with respect to this activity. Investigation of EGCg uptake of liposomal membranes 6) and ultra-structure change on sterilization of trychophyton employing electron microscopy reveals that the action is based on the membrane injury action of bacterial cells. 7) Moreover, it has been reported that the bactericidal effect of EGCg is stronger for gram-positive bacteria than for gram-negative bacteria due to the difference in the amount of EGCg absorbed by the bacterial cell. EGCg was thought to carry a net negative charge in aqueous solution (pH 6-7); furthermore, it was believed to bind to the membrane component of bacteria as a result of electrostatic interaction, which led to membrane damage. 8)However, chemical elucidation of the mechanism via which EGCg causes membrane injury following binding to the liposome remains unclear. We previously established a highly sensitive analytical method for catechin using peroxalate chemiluminescent reaction.9) The principle is based on the reaction mechanism in which catechin reacts with dissolved oxygen in basic solution, resulting in the generation of active oxygen. In this study, we established that the reactive oxygen produced by catechin was hydrogen peroxide via chemiluminescent methodology and electron spin resonance (ESR) using a spin-trapping method. We believed that the hydrogen peroxide generation ability of EGCg was a central component with respect to bactericidal activity; consequently, examination was conducted in detail regarding the role of hydrogen peroxide-mediated bactericidal activity of EGCg and the effect of bactericidal activity of EGCg and hydrogen peroxide in terms of gram-negative (9 o...

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