1 Department of Microbiology and Immunology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 1428555; 2 Tokyo Food Techno Co., Ltd, Tokyo, Japan
Received 23 July 2001; returned 27 December 2001; revised 17 July 2002; accepted 9 September 2002
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Abstract |
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Introduction |
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Vancomycin is an inhibitor of bacterial cell wall synthesis and is the first choice for MRSA treatment. Nevertheless, there are increasing reports of vancomycin-resistant MRSA.8,9 Here we present an extension of this work to look at the interaction between EGCg and vancomycin, as well as an extended range of other non-cell wall active antibiotics.
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Materials and methods |
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EGCg was extracted from green tea and the purity was 98%, as confirmed by high-performance liquid chromatography. The following antibiotics were purchased from the sources indicated: minocycline, streptomycin, kanamycin, erythromycin, rifampicin, ofloxacin and vancomycin (Sigma, St Louis, MO, USA); tetracycline, gentamicin, chloramphenicol and polymyxin B (Wako Pure Chemical Industries, Tokyo, Japan); teicoplanin (Aventis Pharma Japan, Tokyo, Japan).
Bacterial strains
Eight clinical isolates of MRSA were from specimens submitted for routine culture at the clinical microbiology laboratories of Showa University Hospital. All the strains were identified by PCR analysis of mecA gene expression as reported previously.3 Escherichia coli ATCC 25922 was used to test the EGCgpolymyxin B combination. The antimicrobial assay was carried out in MuellerHinton broth (MHB, Becton Dickinson, Cockeysville, MD, USA) supplemented with Ca2+ 25 mg/L and Mg2+ 12.5 mg/L.
MIC determination
MICs were determined using the broth microdilution method at a final inoculum of >5 x 105 cfu/mL according to the guidelines of the NCCLS.10 After incubation at 35°C for 24 h, the lowest concentration of the two-fold serially diluted antibiotic(s) at which no visible growth occurred was defined as its MIC. The growth inhibition was also carried out in 3 mL of MHB with a start inoculum of 5 x 105 cfu/mL and the bacterial growth was detected using a spectrophotometer (OD at 600 nm). Combination effects were confirmed by the chequerboard method. Two-fold serial dilutions of an antibiotic were tested in combination with two-fold serial dilutions of EGCg.
Criteria to evaluate the combination effects between EGCg and antibiotics
The combination effects were evaluated by a fractional inhibitory concentration (FIC) index. FIC was calculated as the MIC of antibiotic and EGCg in combination, divided by the MIC of the antibiotic or EGCg alone, and the FIC index was obtained by adding the FICs. FIC indices were interpreted as synergic when values were 0.5 and as antagonistic when values were >4. If a combination with EGCg resulted in a reduction in the antibacterial activity of an antibiotic, but the FIC index was still <4, it was defined as an antagonistic tendency. The results between synergy and antagonistic tendency were defined as additive or indifferent.
Data presentation
The experiments were repeated three times for each strain. The combination effects between the antibiotics and EGCg for all eight strains of MRSA were determined and the eight strains did show the same tendencies. The data for strain F-74 only are presented as representative of the eight strains.
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Results |
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Figure 1i shows the chemical structure of EGCg. The MIC of EGCg alone for MRSA strains was 100 mg/L. EGCgvancomycin and EGCgteicoplanin combinations showed an unexpected antagonistic tendency at certain concentrations and ratios. For example, in Figure 1b, EGCg at 12.5 mg/L antagonized the activity of teicoplanin at 0.5 mg/L, but EGCg at 6.25 and 25 mg/L showed an additive effect with teicoplanin at the same concentration. The antagonistic tendency was clearer when the bacteria were cultured at a fast-growing condition (shaking at 200 rpm) for 8 h or at an early phase of stationary culture. The antagonistic tendency was further confirmed by counting the cfu after plating the cells on agar plates (data not shown). Combination of EGCg with polymyxin B was also tested against E. coli. EGCg apparently antagonized the activity of polymyxin B (Figure 1c). The MIC of polymyxin B increased from 0.25 to 2 mg/L in the presence of 12.5 mg/L EGCg. The FIC index was >4.
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Additive or indifferent effects were observed in combinations of EGCg with tetracycline, minocycline, chloramphenicol, gentamicin, streptomycin, kanamycin, erythromycin, ofloxacin and rifampicin. The partial data are shown in Figure 1(dh).
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Discussion |
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The additive or indifferent interactions were observed between EGCg and the inhibitors of either protein or nucleic acid synthesis. The increased permeability of the cell wall/membrane to antibiotics caused by the EGCg-induced damage of the cell wall might be the main factor for the additive effects.
The synergic effects between EGCg and ß-lactams suggest a possible clinical use of EGCg to treat MRSA-infected patients, especially with topical or digestive tract infections. When we attempted to enhance the antibacterial activities of vancomycin and teicoplanin against MRSA through combinations with EGCg, however, an antagonistic tendency between EGCg and the two antibiotics was observed. According to the mechanism of synergy between EGCg and ß-lactams against MRSA,3 the two antibiotics should have shown synergy with EGCg. Vancomycin and teicoplanin are glycopeptide antibiotics and EGCg may bind directly to the peptide structure of the antibiotics and then interfere with their activities. The balances between the synergic effect and the reduction of the antibacterial activities owing to their binding to each other may result in the antagonistic tendency at certain ratios of the antibiotics and EGCg but additive or indifferent effects at the other ratios (Figure 1b). To confirm this hypothesis, we detected the effect of an EGCgpolymyxin B combination against E. coli. Polymyxin B is also a glycopeptide antibiotic and highly active in vitro against most Gram-negative bacilli. EGCg clearly antagonized the activity of polymyxin B (Figure 1c, FIC index > 4), indicating direct binding between EGCg and glycopeptide antibiotics. Compared with the MIC of EGCg for S. aureus (100 mg/L), the MIC of EGCg for E. coli was >800 mg/L and there was no synergy between EGCg and ampicillin for E. coli.3 The lack of synergy between polymyxin B and EGCg for E. coli may explain the phenomenon that EGCg showed the strongest antagonism to the activity of polymyxin B.
Tea catechins, including EGCg, structurally belong to plant polyphenols and are generally known as tannins, as reviewed previously.11,12 EGCg at concentrations >50 mg/L did precipitate proteins in broth, indicating a direct binding of EGCg with glycoproteins or polypeptides. The common property of tannins also strongly supports our explanation for the antagonistic tendency between EGCg and glycopeptide antibiotics.
It is hard to predict either synergic or antagonistic effects in vivo just according to the in vitro evidence presented. Usually, EGCg concentration in tea beverage is 23 g/L. Compared with that, 6.25, 12.5 or 25 mg/L EGCg are low concentrations. However, it is difficult to estimate the concentration of bio-available EGCg in vivo after drinking tea or taking EGCg capsules. EGCg is absorbed through the digestive tract and distributed to many organs of animals and humans. In rat blood plasma, EGCg at 5.6 mg/L was detected after being orally administered with EGCg at 500 mg/kg body weight,13 and total catechins at 15112 mg/L were detected 2 h after being orally administered with catechins at 5 g/kg body weight.14 In human blood plasma, EGCg at 2 mg/L was detected after 90 min of taking 525 mg EGCg capsules.15 Therefore, tea and EGCg may affect the activities of antibiotics not only in vitro but also in vivo.
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Footnotes |
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References |
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