Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, W.-H. Articles by Shimamura, T. Search for Related Content PubMed PubMed Citation Articles by Zhao, W.-H. Articles by Shimamura, T.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, July 2002, p. 2266-2268, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2266-2268.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inhibition of Penicillinase by Epigallocatechin Gallate Resulting in Restoration of Antibacterial Activity of Penicillin against Penicillinase-Producing Staphylococcus aureus

Wei-Hua Zhao,^1* Zhi-Qing Hu,¹ Yukihiko Hara,² and Tadakatsu Shimamura¹

Department of Microbiology and Immunology, Showa University School of Medicine,¹ Tokyo Food Techno Co., Ltd., Tokyo, Japan²

Received 11 January 2002/ Returned for modification 11 March 2002/ Accepted 10 April 2002

Top Abstract Text References

 
The combination of epigallocatechin gallate (EGCg, a main constituent of tea catechins) with penicillin showed synergism against 21 clinical isolates of penicillinase-producing Staphylococcus aureus. Besides binding directly to peptidoglycan, the inhibition of penicillinase activity by EGCg is responsible for the synergism. EGCg inhibited the penicillinase activity in a dose-dependent fashion, with a 50% inhibitory concentration of 10 µg/ml.

Top Abstract Text References

 
The emergence and spread of bacterial strains producing ß-lactamase have greatly diminished the usefulness of ß-lactams (12). Even ß-lactamase-stable ß-lactams can be slowly hydrolyzed by staphylococcal ß-lactamases (8), and overproduction of ß-lactamases could result in borderline resistance (11, 16). The combinations of ß-lactams and ß-lactamase inhibitors (such as sulbactam, clavulanic acid, and tazobactam) are a successful strategy to overcome infections caused by bacteria producing ß-lactamases (9). But the recent emergence of bacterial strains producing inhibitor-resistant enzymes could be related to the frequent use of clavulanate (1, 2, 17). Furthermore, the appearance of extended-spectrum ß-lactamases and IMP-1 (a new ß-lactamase) is now threatening the value of broad-spectrum cephalosporins and carbapenems against various bacterial infections (4, 7, 18). It seems that the introduction of any new ß-lactam will be followed by the appearance of a new ß-lactamase. Therefore, any strategy to prevent inactivation of ß-lactams by ß-lactamases is of particular significance.

We recently confirmed that epigallocatechin gallate (EGCg, a main constituent of tea catechins) enhances the activity of ß-lactams against methicillin-resistant Staphylococcus aureus owing to interference with the integrity and biosynthesis of the bacterial cell wall through direct binding to peptidoglycan (19). In this report, we present in vitro evidence that EGCg inhibits penicillinase activity, thus restoring the antibacterial activity of penicillin against penicillinase-producing S. aureus.

EGCg with a purity of 98% was extracted from green tea. Penicillin, ampicillin, and purified penicillinase from Bacillus cereus (Sigma, St. Louis, Mo.), nitrocefin (Oxoid, Basingstoke, United Kingdom), and potassium clavulanate (Wako Pure Chemical Industries, Osaka, Japan) were used.

Twenty-one clinical isolates of penicillinase-producing S. aureus were from the Clinical Microbiology Laboratories of Showa University Hospital. All the strains were identified by PCR analysis for the presence of blaZ gene as reported previously (10). The production of ß-lactamase was tested with a nitrocefin assay (15). Two standard strains, S. aureus ATCC 25923 and Escherichia coli ATCC 25922, were used as controls. Mueller-Hinton broth (MHB) supplemented with 25 mg of Ca²⁺/liter and 12.5 mg of Mg²⁺/liter was used. The MICs and fractional inhibitory concentration (FIC) indices were determined by the broth microdilution and checkerboard methods (5, 6). Synergy between penicillin and EGCg was indicated by an FIC index of 0.5.

To prepare the cell-free supernatant of penicillinase, S. aureus 226, a strain producing high levels of penicillinase even without any inducer, was cultured in MHB at 35°C to an optical density at 600 nm of 0.4. After centrifugation and filtration, the cell-free supernatant was collected as the crude extract of penicillinase. The supernatant contained about 0.015 U of penicillinase per ml, as confirmed by a nitrocefin assay.

To confirm the protection of penicillin or ampicillin from penicillinase by EGCg, the penicillin-susceptible S. aureus strain ATCC 25923 (5 x 10⁴ cells) was inoculated in 100 µl of the cell-free supernatant of penicillinase in the presence of various concentrations of penicillin and EGCg. The ampicillin-susceptible strain E. coli ATCC 25922 (5 x 10⁴ cells) was inoculated in 100 µl of MHB containing various concentrations of ampicillin and EGCg as well as the purified penicillinase. After culture at 35°C for 24 h (S. aureus) or 18 h (E. coli), the MICs were determined. Changes in penicillin and ampicillin MICs were considered evidence of EGCg-induced protection of penicillin and ampicillin from penicillinase.

To prove the direct inhibition of penicillinase by EGCg, the purified penicillinase (10 U/ml) was preincubated in 100 µl of MHB with various concentrations of EGCg for 30 min and 18 h at 35°C in a 96-well culture plate. Nitrocefin (250 µg/ml) was then added as the substrate. ß-Lactamase broke down the ring of nitrocefin, resulting in a color change from yellow to dark red. The color changes were recorded by detecting the optical density at 492 of each well at 10 min with a spectrophotometer. The concentration of EGCg required for 50% inhibition (IC₅₀) of the enzyme activity was determined graphically according to the standard curve of penicillinase under the same assay conditions.

The MICs and FIC indices of penicillin in combination with EGCg against the 21 clinical isolates are presented in Table 1. The MIC range of penicillin was 2 to 128 µg/ml (the susceptibility breakpoint was 0.125 µg/ml). The MIC of EGCg was 100 µg/ml, and EGCg alone did not enhance the production of penicillinase. Synergism occurred against 48, 81, and 100% of the tested strains in combinations of penicillin with EGCg at 3.125, 6.25, and 12.5 µg/ml, respectively. The MIC at which 50% of strains were inhibited (MIC₅₀) of penicillin decreased from 32 µg/ml to 8, 8, 2, and 0.5 µg/ml and the MIC₉₀ decreased from 128 µg/ml to 64, 32, 16, and 8 µg/ml in the presence of EGCg at 3.125, 6.25, 12.5, and 25 µg/ml, respectively. The synergistic bactericidal activity of the penicillin-EGCg combination was also observed in a time-kill assay (data not shown).

View this table: [in this window] [in a new window]

TABLE 1. MICs and FIC indices of penicillin in combination with EGCg against 21 isolates of penicillinase-producing S. aureusa

The MIC of penicillin for S. aureus ATCC 25923 rose from 0.125 to 512 µg/ml in the cell-free supernatant containing penicillinase. EGCg blocked the penicillinase activity in a dose-dependent manner and thus restored the MICs of penicillin from 512 µg/ml to 256, 64, 8, and 0.125 µg/ml at concentrations of 3.125, 6.25, 12.5, and 25 µg/ml, respectively (Fig. 1A).

View larger version (22K): [in this window] [in a new window]

FIG. 1. Protection of penicillin (A) and ampicillin (B) from penicillinase by EGCg. (A) Penicillin-susceptible S. aureus ATCC 25923 cells were inoculated in the cell-free supernatant containing about 0.015 U of penicillinase (PCase) per ml in the presence of twofold serial dilutions of penicillin and EGCg. (B) Ampicillin-susceptible E. coli ATCC 25922 cells were inoculated in MHB containing purified penicillinase at 0.001, 0.005, and 0.01 U/ml in the presence of twofold serial dilutions of ampicillin and EGCg. After incubation at 35°C for 24 h (S. aureus) and 18 h (E. coli), the MICs were determined.

Similarly, the MICs of ampicillin for E. coli ATCC 25922 rose from 8 µg/ml to 128, 1,024, and 2,048 µg/ml in the presence of the purified penicillinase at 0.001, 0.005, and 0.01 U/ml, respectively. EGCg protected the antibacterial activity of ampicillin. In the presence of 0.01 U of penicillinase per ml, for example, the MICs of ampicillin were restored from 2,048 µg/ml to 1,024, 64, and 16 µg/ml by EGCg at 3.125, 6.25, and 12.5 µg/ml, respectively (Fig. 1B). The direct effect of EGCg against E. coli can be omitted, because the MIC of EGCg was more than 800 µg/ml and there was no synergism between EGCg and ampicillin against E. coli (19).

Figure 2 shows that EGCg directly inhibited the activity of penicillinase in a dose-dependent manner. The IC₅₀s of EGCg were 10 µg/ml (21 µM) and 44 µg/ml (96 µM) for the 18-h and 30-min preincubations, respectively. The IC₅₀s of clavulanic acid, a control inhibitor run under the same assay conditions, were 2.5 µg/ml (10.5 µM) and 13.5 µg/ml (56 µM), respectively.

View larger version (19K): [in this window] [in a new window]

FIG. 2. Direct inhibition of penicillinase activity by EGCg. Purified penicillinase (10 U/ml) was incubated with EGCg in 100 µl of MHB at 35°C for 18 h prior to the addition of nitrocefin as its substrate. The optical density at 492 nm was then determined with a spectrophotometer.

The above results demonstrated that besides the effect of EGCg on the cell wall, the direct inhibition of penicillinase activity by EGCg is responsible for synergism. EGCg destroys the penicillinase activity, protecting penicillin or ampicillin from inactivation.

The safe consumption of tea for thousands of years indicates the low toxicity of tea and EGCg. EGCg is absorbed through the digestive tract and distributed to many organs in animals and humans (3, 13, 14). EGCg at 5.6 µg/ml in rat blood plasma was detected after administration of 500 mg/kg of body weight (13). EGCg at 2 µg/ml in human blood plasma was detected 90 min after 525-mg EGCg capsules were taken (14). In this experiment, potent synergy against 48, 81, and 100% of the tested isolates was observed with the combination of penicillin and EGCg at 3.125, 6.25, and 12.5 µg/ml, respectively, indicating that a bioavailable concentration with in vivo activity may be achievable.

 
We thank Rika Wakuta and Kunihide Gomi, Central Clinical Laboratories, Showa University Hospital, for providing S. aureus isolates.

This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Phone: 81-3-3784-8131. Fax: 81-3-3784-3069. E-mail: whzhao{at}med.showa-u.ac.jp.

Top Abstract Text References

 

1
1. Blasquez, J., M.-R. Baquero, I. Canton, I. Alos, and F. Baquero. 1993. Characterization of a new TEM-type ß-lactamase resistant to clavulanate, sulbactam, and tazobactam. Antimicrob. Agents Chemother. 37:2059-2063.[Abstract/Free Full Text]
2
2. Chaibi, E. B., D. Sirot, G. Paul, and R. Labia. 1999. Inhibitor-resistant TEM ß-lactamase: phenotypic, genetic and biochemical characteristics. J. Antimicrob. Chemother. 43:447-458.[Abstract/Free Full Text]
3
3. Chen, L., M. J. Lee, H. Li, and C. S. Yang. 1997. Absorption, distribution, elimination of tea polyphenols in rats. Drug Metab. Dispos. 25:1045-1050.[Abstract/Free Full Text]
4
4. Heritage, J., F. H. M'Zali, D. Gascoyne-Binzi, and P. M. Hawkey. 1999. Evolution and spread of SHV extended-spectrum ß-lactamases in Gram-negative bacteria. J. Antimicrob. Chemother. 44:309-318.[Abstract/Free Full Text]
5
5. Hu, Z.-Q., W.-H. Zhao, N. Asano, Y. Yoda, Y. Hara, and T. Shimamura. 2002. Epigallocatechin gallate synergistically enhances the activity of carbapenems against methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:558-560.[Abstract/Free Full Text]
6
6. Hu, Z.-Q., W.-H. Zhao, Y. Hara, and T. Shimamura. 2001. Epigallocatechin gallate synergy with ampicillin-sulbactam against 28 clinical isolates of methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 48:361-364.[Abstract/Free Full Text]
7
7. Ito, H., Y. Arakawa, S. Ohsuka, R. Wacharotayankun, N. Kato, and M. Ohta. 1995. Plasmid-mediated dissemination of the metallo-ß-lactamase gene bla_IMP among clinically isolated strains of Serratia marcescens. Antimicrob. Agents Chemother. 39:824-829.[Abstract/Free Full Text]
8
8. Kernodle, D. S., C. S. Stratton, L. W. McMurray, J. R. Chipley, and P. A. McGraw. 1989. Differentiation of beta-lactamase variants in Staphylococcus aureus by substrate hydrolysis profiles. J. Infect. Dis. 159:103-108.[Medline]
9
9. Maddux, M. 1991. Effects of ß-lactamase-mediated antimicrobial resistance: the role of ß-lactamase inhibitors. Pharmacotherapy 11(Suppl.):40-50.
10
10. Martineau, F., F. J. Picard, L. Grenier, P. H. Roy, M. Ouellette, the ESPRIT Trial, and M. G. Bergeron. 2000. Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. J. Antimicrob. Chemother. 46:527-533.[Abstract/Free Full Text]
11
11. McDougal, L. K., and C. Thornsberry. 1986. The role of ß-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins. J. Clin. Microbiol. 23:832-839.[Abstract/Free Full Text]
12
12. Medeiros, A. A. 1984. Beta-lactamases. Br. Med. Bull. 40:18-27.[Free Full Text]
13
13. Nakagawa, K., and T. Miyazawa. 1997. Absorption and distribution of tea catechin, (-)-epigallocatechin-3-gallate, in rats. J. Nutr. Sci. Vitaminol. (Tokyo) 43:679-684.
14
14. Nakagawa, K., S. Okuda, and T. Miyazawa. 1997. Dose-dependent incorporation of tea catechins, (-)-epigallocatechin-3-gallate and (-)-epigallocatechin, into human plasma. Biosci. Biotechnol. Biochem. 61:1981-1985.[Medline]
15
15. O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. Shingler. 1972. Novel method for detection of ß-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288.[Abstract/Free Full Text]
16
16. Tomasz, A., H. B. Drugeon, H. M. de Lencastre, D. Jabes, L. McDougal, and J. Bille. 1989. New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob. Agents Chemother. 33:1869-1874.[Abstract/Free Full Text]
17
17. Vedel, G., A. Belaaouaj, G. Lilly, R. Labia, A. Philippon, P. Nevot, and G. Paul. 1992. Clinical isolates of Escherichia coli producing TRI ß-lactamases: novel TEM-enzymes conferring resistance to ß-lactamase inhibitors. J. Antimicrob. Chemother. 30:449-462.[Abstract/Free Full Text]
18
18. Watanabe, M., S. Iyobe, M. Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151.[Abstract/Free Full Text]
19
19. Zhao, W-H., Z.-Q. Hu, S. Okubo, Y. Hara, and T. Shimamura. 2001. Mechanism of synergy between epigallocatechin gallate and ß-lactams against methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 45:1737-1742.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, July 2002, p. 2266-2268, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2266-2268.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Zhao, W.-H., Hu, Z.-Q., Shimamura, T. (2008). Potency of IMP-10 metallo-{beta}-lactamase in hydrolysing various antipseudomonal {beta}-lactams. J Med Microbiol 57: 974-979 [Abstract] [Full Text]  
• Blanco, A. R., Sudano-Roccaro, A., Spoto, G. C., Nostro, A., Rusciano, D. (2005). Epigallocatechin Gallate Inhibits Biofilm Formation by Ocular Staphylococcal Isolates. Antimicrob. Agents Chemother. 49: 4339-4343 [Abstract] [Full Text]  
• Sudano Roccaro, A., Blanco, A. R., Giuliano, F., Rusciano, D., Enea, V. (2004). Epigallocatechin-Gallate Enhances the Activity of Tetracycline in Staphylococci by Inhibiting Its Efflux from Bacterial Cells. Antimicrob. Agents Chemother. 48: 1968-1973 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, W.-H. Articles by Shimamura, T. Search for Related Content PubMed PubMed Citation Articles by Zhao, W.-H. Articles by Shimamura, T.