Epigallocatechin gallate synergy with ampicillin/sulbactam against 28 clinical isolates of methicillin-resistant Staphylococcus aureus

Zhi-Qing Hua,*, Wei-Hua Zhaoa, Yukihiko Harab and Tadakatsu Shimamuraa

a Department of Microbiology and Immunology, Showa University School of Medicine; b Tokyo Food Techno Co., Ltd, Tokyo, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Compared with ampicillin, oxacillin, cefmetazole and imipenem, the combination of ampicillin and sulbactam at a constant ratio of 2:1 showed the greatest effect against 28 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), but MICs of ampicillin/sulbactam were still above the resistance breakpoint. When ampicillin/sulbactam was further combined with epigallocatechin gallate (EGCg, a main constituent of tea catechins), the MIC90 of ampicillin/ sulbactam was reduced to 4 mg/L, the susceptibility breakpoint. The fractional inhibitory concentration indices were between 0.19 and 0.56 in combination with 6.25 and 25 mg/L EGCg, respectively, indicating that ampicillin/sulbactam and EGCg combination may be effective against MRSA infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ampicillin has good bactericidal activity; however, it can be degraded by ß-lactamases produced by various bacteria, including some of the methicillin-susceptible Staphylococcus aureus strains (MSSA) and most of the methicillin-resistant strains (MRSA). Sulbactam is a ß-lactamase inhibitor and acts primarily by irreversible inactivation of ß-lactamases. The combination of ampicillin/sulbactam inhibits ß-lactamase activity and increases the spectrum of activity. According to previous reports and our in vitro experiments, the combination is effective, but is not powerful enough against MRSA, including strains producing ß-lactamase.1,2

Epigallocatechin gallate (EGCg; a main constituent of tea catechins) is synergic with ß-lactams against MRSA.35 We hypothesized that ampicillin/sulbactam could be effective against MRSA if combined with EGCg.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics and EGCg

Nitrocefin was purchased from Oxoid (Basingstoke, UK). Oxacillin, methicillin, ampicillin, imipenem, cefmetazole and ampicillin/sulbactam were purchased from Sigma (St Louis, MO, USA). EGCg was extracted from green tea with a purity of 98% as confirmed by high-performance liquid chromatography analysis.

Bacterial strains and media

Twenty-eight clinical isolates of MRSA were obtained from Fujigaoka and Hatanodai Hospitals of Showa University. MSSA ATCC 25923 and ß-lactamase-producing MSSA 226 were used as controls. Cation-adjusted Mueller– Hinton broth and agar (Becton Dickinson, Cockeysville, MD, USA) were used for susceptibility tests. Heart infusion broth (HIB; Difco Laboratories, Detroit, MI, USA) was used for ß-lactamase tests.

ß-Lactamase tests and mecA gene detection

For ß-lactamase tests, an inoculum of 104 bacteria/well was incubated at 35°C overnight in 100 µL HIB in the absence/ or presence of 0.5 mg/L methicillin, which was used to induce the ß-lactamase production. Fifty microlitres of nitrocefin 500 mg/L was then added to the cultures. ß-Lactamase broke down the ring of nitrocefin resulting in a colour change from yellow to red. The colours were graded from no change (–) to dark red (3+) at 5, 30, 60 and 120 min, respectively. All the strains were also identified by PCR analysis for mecA gene expression as reported previously.6

Susceptibility tests

MICs were determined using the broth dilution and chequerboard methods at a final inoculum >5 x 105 cfu/mL as reported previously.6 Ampicillin/sulbactam combination was tested at a constant ratio of 2:1 as used in the clinically available formulation. MICs of ampicillin/sulbactam were presented as the concentrations of ampicillin. Synergy between ampicillin/sulbactam and EGCg was evaluated by a fractional inhibitory concentration (FIC) index. FIC was calculated as the MIC of ampicillin/sulbactam EGCg in combination divided by the MIC of ampicillin/sulbactam or EGCg alone, and the FIC index was obtained by adding the FICs. If an FIC index was <=0.5, the combination was defined as synergic. Synergy was also determined by time– kill assay and defined as a reduction of >=2 log10 of the initial inoculum (106 cfu/mL) at 24 h by the combination of ampicillin/sulbactam and EGCg at sub-MICs. The time– kill assays were performed in triplicate and the data presented are mean ± s.d.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All 28 isolates were resistant to the tested ß-lactams, except for F-98, which was susceptible to imipenem. Sixty-eight per cent (19/28) of the strains produced ß-lactamase. Compared with ampicillin, oxacillin, cefmetazole and imipenem, ampicillin/sulbactam showed the strongest anti-MRSA effect, especially against the ß-lactamase-producing strains of MRSA and MSSA 226. The MIC range of ampicillin against ß-lactamase-producing MRSA changed from 64–256 to 16–32 mg/L in the presence of sulbactam, but no change was observed against ß-lactamase-non-producing MRSA. Nevertheless, MICs of ampicillin/sulbactam were still above its resistant breakpoint (>=16 mg/L). EGCg also showed anti-MRSA effect with an MIC at 100 mg/L. When ampicillin/sulbactam at sub-MICs was further combined with EGCg at sub-MICs, potent synergy and dose-dependent reversion of the resistance against ampicillin/sulbactam were observed (TableGo). The MIC50 of ampicillin/sulbactam decreased to 8 mg/L in the presence of 6.25 mg/L EGCg, and the MIC90 of ampicillin/sulbactam decreased to 4 mg/L in the presence of 25 mg/L EGCg. The synergic effect between ampicillin/sulbactam and EGCg was very similar for the ß-lactamase-producing and ß-lactamase-non-producing MRSA isolates.


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Table. MICs of ß-lactams and FIC indices of ampicillin/sulbactam in combination with EGCg against 28 isolates of MRSA
 
Time–kill curves show the synergic bactericidal effects of ampicillin/sulbactam and EGCg against two typical strains of the ß-lactamase-producing and ß-lactamase-non-producing isolates (FigureGo). The combinations resulted in a reduction of >=2 log10 of the initial inoculum at 24 h.



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Figure. Time–kill curves of the synergic activity of the combination of ampicillin/sulbactam and EGCg. (a) F-10, ß-lactamase-producing MRSA. (b) F-56, ß-lactamase-non-producing MRSA. {circ}, no addition; {triangledown}, ampicillin/sulbactam alone at 0.5 x MIC (8 mg/L for F-10 and 16 mg/L for F-56); {square}, EGCg alone at 25 mg/L (0.25 x MIC); •, EGCg at 6.25 mg/L plus ampicillin/sulbactam at 0.5 x MIC; {blacktriangledown}, EGCg at 25 mg/L plus ampicillin/sulbactam at 0.25 x MIC.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although MRSA is resistant to almost all ß-lactams, ampicillin in combination with ß-lactamase inhibitors may be effective, because most MRSA produce ß-lactamase, and ampicillin has a relative affinity for PBP2' 10–15 times greater than that of penicillinase-resistant ß-lactams.7 Recently, an ampicillin and sulbactam combination (UNASYN) has been approved by US Food and Drug Administration for intravenous administration in the treatment of various infections.

About 10–15% of the dry weight of green tea is made up of catechins, including catechin, epicatechin, epigallocatechin, epicatechin gallate and EGCg. EGCg, the main constituent (60%) of tea catechins, has the best antibacterial activity. Interestingly, catechins demonstrate synergy with ß-lactams for MRSA.35 We recently confirmed that EGCg is synergic with ß-lactams owing to interference with the integrity and biosynthesis of the bacterial cell wall through direct binding to peptidoglycan.6

The potent synergy between ampicillin/sulbactam and EGCg suggests a possible clinical use for MRSA infection. Tea, one of the most popular beverages, is consumed every day by billions of people. Capsules of tea catechins and EGCg are also becoming available for research and pre-clinical trial. Usually, EGCg concentration in tea beverage is 2–3 g/L. EGCg is absorbed through digestive tract and distributed to many organs of animals and humans.810 In rat blood plasma, EGCg at 5.6 mg/L was detected after oral administration of EGCg at 500 mg/kg body weight,8 and total catechins at 15–112 mg/L were detected at 2 h after oral dosing with catechins at 5 g/kg body weight.9 In human blood plasma, EGCg at 2 mg/L was detected 3 h after taking 525 mg EGCg capsules.10 Therefore, further experiments are needed to confirm the possibility of using ampicillin/sulbactam and EGCg combination against MRSA infections.


    Notes
 
* Corresponding author. Tel: +81-3-3784-8131; Fax: +81-3-3784-3069; E-mail: zqhu{at}med.showa-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Backo, M., Gaenger, E., Burkart, A., Chai, Y. L. & Bayer, A. S. (1999). Treatment of experimental staphylococcal endocarditis due to a strain with reduced susceptibility in vitro to vancomycin: efficacy of ampicillin–sulbactam. Antimicrobial Agents and Chemotherapy 43, 2565–8.[Abstract/Free Full Text]

2 . Barry, A. L. & Jones, R. N. (1990). In vitro activities of ampicillin– sulbactam and cefoperazone–sulbactam against oxacillin-resistant staphylococci. Antimicrobial Agents and Chemotherapy 34, 1830–2.[ISI][Medline]

3 . Takahashi, A., Cai, Z., Toda, M., Hara, Y. & Shimamura, T. (1995). Appearance of antibacterial activity of oxacillin against methicillin-resistant Staphylococcus aureus (MRSA) in the presence of catechin (in Japanese). Journal of the Japanese Association for Infectious Diseases 69, 1126–34.[Medline]

4 . Yamazaki, K. (1996). Enhancing effect of Japanese green tea extract on the growth-inhibitory activity of antibiotics against isolated MRSA strains (in Japanese). Japanese Journal of Chemotherapy 44, 477–82.

5 . Yam, T. S., Hamilton-Miller, J. M. T. & Shah, S. (1998). The effect of a component of tea (Camellia sinensis) on methicillin resistance, PBP2' synthesis, and ß-lactamase production in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 42, 211–6.[Abstract]

6 . Zhao, W.-H., Hu, Z.-Q., Okubo, S., Hara, Y. & Shimamura, T. (2001). Mechanism of synergy between epigallocatechin gallate and ß-lactams against methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 45, 1737–42.[Abstract/Free Full Text]

7 . Franciolli, M., Bille, J., Glauser, M. P. & Moreillon, P. (1991). ß-Lactam resistance mechanisms of methicillin-resistant Staphylococcus aureus. Journal of Infectious Diseases 163, 514–23.[ISI][Medline]

8 . Nakagawa, K. & Miyazawa, T. (1997). Absorption and distribution of tea catechin, (–)-epigallocatechin-3-gallate in rats. Journal of Nutritional Science and Vitaminology (Tokyo) 43, 679–84.[ISI][Medline]

9 . Zhu, M., Chen, Y. & Li, R. C. (2000). Oral absorption and bioavailability of tea catechins. Planta Medica 66, 444–7.[ISI][Medline]

10 . Nakagawa, K., Okuda, S. & Miyazawa, T. (1997). Dose-dependent incorporation of tea catechins, (–)-epigallocatechin-3-gallate and (–)-epigallocatechin, into human plasma. Bioscience Biotechnology and Biochemistry 61, 1981–5.[ISI][Medline]

Received 2 February 2001; returned 5 June 2001; revised 20 June 2001; accepted 25 June 2001