1518

Antibacterial Activity of Epigallocatechin Gallate against Methicillin-Resistant Staphylococcus aureus

Kenji KONO1), Ichiro TATARAn, Seiji TAKEDA1), Kikuo ARAKAWA1) and Yukihiko HARA2) 1) The Second Department of Internal Medicine, Fukuoka University, School of Medicine 2)Mitsui Norin Co ., Ltd. Food Research Laboratories, Fujieda, Shizuoka 426, Japan (Received: September 14, 1994) (Accepted: October 26, 1994)

Key words: MRSA, green tea, catechin, epigalocatechin gallate

Abstract

The antibacterial activity of epigallocatechin gallate (EGCg) , a catechin, against 53 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) was evaluated and expressed as minimal inhibitory concentration (MIC). The MIC50 and MIC90 of the strains were found be 64 and 126

g/ml, respectively by the microdilution method. A time-kill study using an isolate showed that EGCgƒÊ

appeared to be bacteriostatic at 1-2 x MIC and bactericidal at 6 x MIC against MRSA . In addition,

the activity of EGCg was stable to various physical conditions including boiling or freezing . These findings suggest that EGCg could be a useful agent for treating MRSA infection .

Introduction

It is well known that Japanese green tea has antibacterial activity against various foodborne pathogenic bacteria including Clostridium botulinum 1,2),Staphylococcus aureus3), Vibrio parahemoly - ticus3), Clostridium perfringens 3), and Bacillus cereus 3), Salmonella typhi, Shigella dysenteriae, Campylobacter jejuni, and Vibrio cholerae4). In addition , there are reports that catechin is effective against infections due to Bordetella pertussis5), Mycoplasma pneumoniae6) , Trichophyton species7), rotavirus, enterovirus8 , and influenza virus9). Moreover, the catechin in tea extract inhibits glucan synthesis by glucosyltransferase from Streptococcus mutans , thereby preventing dental caries10) Recently, Toda et al. showed that epigallocatechin gallate (EGCg) , one of the major classes of catechins, has antibacterial activity against methicillin-resistant S. aureus (MRSA)10). MRSA is a major nosocomial pathogen which causes problems worldwide . However, no clinical application of catechin in MRSA infection has yet been reported. The minimum inhibitory concentration (MIC) of catechin against MRSA likewise has not been determined. In order to determine the possible clinical usefulness of catechin in MRSA infections , we quantitatively measurd the activity of extracts of Japanese tea including EGCg against MRSA strains isolated in our university hospital. In addition, we examined the physical effects of conditions such as boiling and freezing, on catechin to decide on appropriate conditions for preservation of catechin for clinical use.

Correspondence to: Kenji KONO The Second Departement of Internal Medicine, School of Medicine , Fukuoka University, 7-45-1, Nanakuma, Jonan-ku, Fukuoka, 814-01 Japan

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Materials and Methods

Bacteria

All strains of S. aureus that were recovered were identified in the Clinical Laboratory Service in our university hospital in 1993 . Those strains of S. aureus that were found to be oxacillin-resistant by the Kirby-Bauer method were considered MRSA. Thus, 53 strains of MRSA were examined in this study. For the study of the time-kill curve and effect of physical treatments on catechin, MRSA strain YK that was isolated from a patient with sepsis was used.

Catechins Purified epigallocatechin galate (EGCg) and Polyphenon-100 (crude extract of Japanese green tea, containing 57% EGCg) (Mitsui Norin Co., Ltd., Fujieda, Shizuoka, Japan) were kindly donated by the Central Laboratory, Mitsui Norin Co.

MICs

All isolates were tested for sensitivity to the antimicrobial agents by the microdilution method to determine the MIC. EGCg and Polyphenon-100 were diluted to concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 ,ag/ml in Mueller-Hinton broth (Difco Laboratories, Detroit, Michigan,

USA). MRSA strains were subcultured overnight, then suspended in saline solution. After transfer of the bacteria in an amount equal to 104 to each well of multiwell culture plates, the plates were incubated at 35•Ž for 18 h. MIC was defined as the lowest concentration of catechin that allowed no visible growth.

Tirne-kill curve

Time-kill studies with catechin were performed with the YK strain of MRSA. The organism was cultured overnight and shaken. After incubation with shaking for 1 h, approximately 106 CFU/ml of bacteria in the log phase were transferred to tubes containing 5 ml of Mueller-Hinton broth. The catechins were added to give final concentrations of 1, 2 and 6 x MIC. The MIC of the YK strain was

64ƒÊg/ml. The tubes were incubsated further at 37•Ž with shaking and samples were withdrawn at

0, 3, 6, and 24 h and spread on agar plates. The numbers of CFU were determined after overnight incubation. Control cultures without catechins were examined in parallel and each experiment was performed in duplicate. Effects of physical conditions on antibacterial activity of catechin

Strain YK was stored in the cold for 40 days, boiled for 30 min in a capped tube to avoid evaporation, and autoclaved 120•Ž for 20 min to determine the effects of these conditions on the antibacterial activity of catechin against MRSA. Before, during, and after these treatments, catechins were tested to determine their MICs against the YK strain.

Results

Table 1 shows the sensitivity of the clinical isolates to each catechin. The MICs of EGCg and

Polyphenon-100 were 32-64ƒÊg/ml and 64-256ƒÊg/ml respectively, against all strains. Fig. 1 shows the

Table 1 Antibacterial activitiy of catechins against 53 clinical isolates of MRSA

*Number of isolates

平成6年12月20日 1520 Kenji KONO et al

Fig. 2 Effects of physical conditions on the antibacterial activity of catechins.

Fig. 1 Bactericidal activity (time-kill curve) of EGCg against MRSA strain YK.

Boiling

Autoclaving

time-kill curve of ECGg against MRSA. In concentrations 1- or 2 times the MIC , the number of bacteria only decreased to 1/10th of the original concentration , suggesting that the activity is bacteriostatic. On the other hand, concentrations 6-fold greater than MIC resulted in 3-log10 decrease

in CFU/ml after 24 h, suggesting that the activity is bactericidal11) . As for the effects of physical conditions on the activity of the catechins (Fig . 2), the MIC of Polyphenon-100, 128ƒÊg/ml, did not increase after refrigeration, freezing, boiling, or autoclaving . The MIC of EGCg was increased, but only from 64 to 128 ,ug/ml (this means one dilution higher by the microdilution method) after refrigeration, freezing, boiling, or autoclaving. In addition , the activity of EGCg and Polyphenon-100 was stable at room temperature for 2 weeks.

Discussion

In our country, Japanese green tea is a traditional , commonly consumed drink which is very familiar to the general population. Catechin is one of the major components of green tea (10-18%)12,13) and is a kind of polyphenol, the chemical structure of which has been determined . EGCg is the most important of the four classes of catechins (comprising about 50%)12,13) , displays various antibacterial activities. The present study revealed the following: 1) Catechins are effective against clinical isolates of MRSA, although the MIC is somewhat higher than that of and-MRSA agents such as vancomycin

(MIC, 1-2ƒÊg/ml). Since the activity of green tea is considered to be due to EGCg and since

感染 症 学 雑 誌 第68巻 第12号 Antibacterial Activity of Catechin against MRSA 1521

Polyphenon-10 consists of 50% EGCg, the results seem to be reasonable. 2) The mechanism of action is killing of the bacteria at high concentrations, suggesting that EGCg has a concentration-dependent bactericidal activity. Toda et al. also showed that EGCg is bactericidal against MRSA14). 3) These compounds are highly stable under various physical conditions including freezing and boiling. In addition, they are preserved well even at room temperature. Thus, catechins are so well preserved that they appear to be candidates for clinical use. However, the MICs of catechin are so high, unlike those of antibiotics, that it may be difficult to achieve catechin tissue levels that are high enough following intestinal absorption after oral administration. Catechins seem to be suitable for oral administration for the treatment of intestinal infections or colonization and for topical administration for the treatment of skin infections or colonization. Inhalation therapy with catechins could also be recommended for respiratory tract infections or colonization. Catechins in solution can be well preserved in a refrigerator for up to a month. In addition, they are heastable, so that even hot water tea extracts (so-called tea drink), which contain antibacterial activity equivalent to 0.5-1.0 mg of polyphenon (data not shown), are also useful when catechin extract is not available, although the concentration does not appear to be high enough for clinical use. In the future, we will investigate the mechanism of action of this EGCg against MRSA using in vitro and in vivo experiments. Furthermore, we may propose a clinical trial of this agent against MRSA infection and colonization.

Acknowledgment

We thank Ms. Sakie Kimura for her technical assistance.

References

1) Hara, Y,,Watanabe, M. & Sakaguchi, G.: The fate of Clostridium botulinum spores inoculated into tea drinks. Nippon Shokuhin Kogyo Gakkaishi 36: 975-979, 1989. 2) Hara, Y. & Watanabe, M.: Antibacterial activity of tea polyphenols against Clostridium botulinum. Nippon Shokuhin Kogyo Gakkaishi 36: 951-955, 1989. 3) Hara, Y. & Ishigami, T.: Antibacterial activities of tea polyphenols against foodborne pathogenic bacteria. Nippon Shokuhin Kogyo Gakkaishi 36: 966-999, 1989. 4) Toda, M., Okubo, S., Ohnishi, R. & Shimamura, T.: Antibacterial and bactericidal activities of Japanese greentea. Jpn. J. Bacteriol. 44: 669-672, 1989. 5) Horiuchi, Y., Toda, M., Okubo, S. et al.: Protective activity of tea and catechins against Bordetella pertussis. J. Jpn. Assoc. Inf. Dis. 66: 599-605, 1992. 6) Chosa, H., Toda, M., Okubo, S., et al.: Antimicrobial and microbicidal activities of tea and catechins against Mycoplasma. J. Jpn. Assoc. Inf. Dis. 66: 606-611, 1992. 7) Okubo, S., Toda, M., Hara, Y. & Shimamura, T.: Antifungal and fungicidal activities of tea extract and catechin against Trichophyton. Jpn. J. Bacteriol. 46, 509-514, 1991. 8) Mukoyama, A., Ushijima, H., Nishimura, S., et al.: Inhibition of rotavirus and enterovirus infections by tea extracts. Jpn. J. Med. Sci. Biol. 44: 181-186, 1991. 9) Nakayama, M., Suzuki, K., Toda, M., Okubo, S., Hara, Y. & Shimamura, T.: Inhibition of the infectivity of influenza virus by tea polyphenols. Antiviral Research, 21: 289-299, 1993. 10) Hattori, M., Kusumoto, I.T., Namba, T., et al.: Effect of tea polyphenol on glucan synthesis by glucosyltransferase from Streptococcus mutans. Chem. Pharm. Bull. 38: 717-720, 1990. 11) National Committee for Clinical Laboratory Standards. Methods for determing bactericidal activity of antimi- crobial agents; Tentative Guideline. NCCLS document M26-T, vol 12 No. 19, 1992. 12) Maeda, S. & Nakagawa, M.: General chemical and physical analysises on various kinds of green tea. Tea Research J. 45: 85-92, 1977 (in Japanese). 13) Hirose, S. & Tamada, S.: A quantitative determination of flavanols in tea by high pressure liquid chromatography. Tea Research J. 50: 51-55, 1979 (in Japanese). 14) Toda, T., Okubo, S., Hara, Y. & Shimamura, T.: Antibacterial and bactericidal activities of tea extracts and catechins against methicilin resistant Staphylococcus aureus. Jpn. J. Bacteriol. 46: 839-845, 1991.

平 成6年12月20日 1522 Kenji KONO et al

メ チ シ リ ン耐 性 黄 色 ブ ドウ球 菌(MRSA)に 対 す る epigallocatechin gallate (EGCg)の 抗 菌 活 性 の検 討

1)福 岡大 学 医学 部 第 二 内科,2)三 井 農林 株 式 会 社食 品総 合研 究 所 向 野 賢 治1)多 々 良 一 郎1)武 田 誠 司1)

荒 川 規 矩 夫1)原 征 彦2)

要 旨 曲線 によ る検 討 で は,EGCgはMRSAに 対 し1 メ チ シ リ ン 耐 性 黄 色 ブ ド ウ 球 菌(MRSA)の 臨 ～2MICで は静菌 的 ,6MICで は殺菌 的に作用 床 分 離53株 に 対 す る epigallocatechin galate す る と思 われた.ま た,EGCgは 煮 沸や凍結 な どの (EGCg)の 抗 菌 活 性 を 最 小 発 育 阻 止 濃 度MICに 物理 学的処理 に対 して も安 定で あった.こ れ らの つ い て 検 討 し た .53株 に 対 す るEGCgの 微 量 液 体 ことか ら,EGCgはMRSA感 染 症 に対 し有 用性 希 釈 法 に よ るMIC50, MIC90は そ れ ぞ れ64,128 が あ ると思われ た. μg/mlで あ っ た.分 離 株1株 を 使 っ た 時 間 殺 菌

感 染 症 学雑 誌 第68巻 第12号