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Planta Med 2003; 69(11): 984-989
DOI: 10.1055/s-2003-45142
Original Paper
Pharmacology
© Georg Thieme Verlag Stuttgart · New York

Theasinensin A, a Tea Polyphenol Formed from (-)-Epigallocatechin Gallate, Suppresses Antibiotic Resistance of Methicillin-Resistant Staphylococcus aureus

Tsutomu Hatano1 , Miwako Kusuda1 , Mami Hori1 , Sumiko Shiota2 , 3 , Tomofusa Tsuchiya2 , Takashi Yoshida1
  • 1Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan
  • 2Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan
  • 3Present address: Department of Microbiology, Faculty of Pharmaceutical Sciences, Shujitu University, Okayama, Japan
Further Information

Dr. T. Hatano

Faculty of Pharmaceutical Sciences

Okayama University

Tsushima

Okayama 700-8530

Japan

Fax: +81-86-251-7936

Email: hatano@pharm.okayama-u.ac.jp

Publication History

Received: April 14, 2003

Accepted: July 12, 2003

Publication Date:
09 January 2004 (online)

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Table of Contents #

Abstract

When (-)-epigallocatechin gallate (EGCG), the main constituent of tea polyphenols, was kept in a neutral buffer, it decomposed rapidly to give theasinensin A as the major product. Theasinensin A suppressed the oxacillin resistance of methicillin-resistant Staphylococcus aureus (MRSA). In the presence of theasinensin A (3.5 × 10 - 5 M), the minimum inhibitory concentrations (MICs) of oxacillin decreased from 256 or 64 μg/mL to 4 μg/mL for the MRSA strains used. The presence of this compound (3.5 × 10 - 5 M) also decreased the MIC of other β-lactam (penicillin G, from 32 μg/mL to 0.125 - 0.5 μg/mL; ampicillin, from 16 - 32 μg/mL to 0.5 - 1 μg/mL) and aminoglycoside (streptomycin, from 4 - 16 μg/mL to 0.125 - 4 μg/mL) antibiotics for the MRSA strains.

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Abbreviations

CFU:colony forming unit

CSMHB:cation-supplemented Mueller-Hinton broth

DETAPAC:diethylenetriaminepentaacetic acid

EGCG:(-)-epigallocatechin gallate

HPLC:high-performance liquid chromatography

MIC:minimum inhibitory concentration

MRSA:methicillin-resistant Staphylococcus aureus

MSSA:methicillin-sensitive Staphylococcus aureus

NMR:nuclear magnetic resonance

PBP2′:penicillin-binding protein 2′

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Key words

Camellia sinensis - Theaceae - epigallocatechin gallate - theasinensin A - gallic acid - polyphenol - MRSA - antibiotic resistance

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Introduction

(-)-Epigallocatechin gallate (EGCG) is the primary polyphenolic constituent of unfermented tea leaves (Camellia sinensis Kuntze, Theaceae). This compound has a protein-binding activity similar to that of tannins despite its low molecular weight [1], and it has potent antioxidant effects [2], [3], [4]. The inhibitory effects on tumor promotion [5], [6] and suppressive effects on the antibiotic resistance of methicillin-resistant Staphylococcus aureus (MRSA) [7], [8] have also been reported. Numerous findings concerning its pharmacological properties prompted studies on its absorption and metabolism after oral administration to mammals, including humans [9], [10], [11], [12], [13].

EGCG and related polyphenols are easily oxidized to a mixture of products [14], [15], [16], [17]. Our present investigation showed that EGCG is unstable, even in solution at a neutral pH. We proposed, therefore, that oxidation products may participate, at least in part, in the effects attributed to EGCG. We isolated several polyphenolic compounds from the reaction mixture obtained upon the treatment of EGCG in neutral solution and examined the effects of these products on MRSA. This paper reports the identification of products from EGCG and the suppressive effect of the primary product, theasinensin A, on the antibiotic resistance of MRSA.

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Materials and Methods

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Materials

EGCG was isolated from tea leaves (Camellia sinensis) [18]. Purity of this compound was estimated using high-performance liquid chromatography (HPLC), where the apparent peak area of this specimen indicated > 99 % of the purity. Diethylenetriaminepentaacetic acid (DETAPAC) and the antibiotics used in the experiments (oxacillin, penicillin G, ampicillin, streptomycin, tetracycline and fosfomycin) were purchased from Sigma. Gallic acid, used for the comparison with the products from EGCG and for the antibacterial assays, was the product of Ishidzu (Osaka, Japan). Erythromycin was from Nacalai (Kyoto, Japan).

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Bacterial strains

MRSA strains, OM481, OM505, OM584 and OM623, were clinical isolates from Okayama University Hospital. The methicillin-sensitive Staphylococcus aureus (MSSA) 209P strain was used as a control. The strains used in this study were maintained in the laboratory of the Department of Microbiology.

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HPLC

Analytical HPLC was performed in an oven at 40 °C using a YMC A302 ODS column (4.6 mm i. d. × 150 mm) with a mixture of 10 mM KH2PO4 - 10 mM H3PO4-acetonitrile (4 : 4 : 2) as an eluant. Detection was by ultraviolet (UV) light absorption at 280 nm.

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Structural change during the incubation of EGCG in solution

A solution of EGCG in a K2HPO4-KH2PO4 buffer (0.1 M, pH 7.4) was kept at 37 °C for 5 h. Chemical changes in the solution were monitored by HPLC. Chemical changes in solutions of different pH values (pH 6.5, 7.0 and 7.8) and the effects of additions of DETAPAC (1.1 mg/mL) and FeCl3 (0.07 mg/mL) on the structure of EGCG were analyzed in analogous ways.

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Isolation of the compounds formed from EGCG

The product mixture in a solution obtained from 100 mg of EGCG in a K2HPO4-KH2PO4 buffer (pH 7.0) was acidified with 5 M HCl to pH 3 and subjected to column chromatography on MCI-gel CHP-20P with increasing concentrations of MeOH in water. Column fractions were further purified by preparative HPLC on a YMC A324 ODS column (10 mm i. d. × 300 mm) with the same solvent system that was used for analytical HPLC, yielding 3.9 mg theasinensin A [19], 2.8 mg theasinensin D [20], 2.8 mg (-)-gallocatechin gallate [21], [22], 2 mg gallic acid, and several other minor unidentified products.

Theasinensin A: [α]D 28: -218° (c 0.27, acetone) {-226.8° (lit. [19])}.

Theasinensin D: [α]D 28: -123° (c 0.12, acetone) {-158.6° (lit. [20])}.

(-)-Gallocatechin gallate: [α]D 28: -30.6° (c 0.094, acetone) {-37° (lit. [22])}.

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Evaluation of minimum inhibitory concentrations (MICs)

A cation-supplemented Mueller-Hinton broth (CSMHB) that contained CaCl2 (50 mg/L) and MgCl2 (25 mg/L) was used as the medium for the MRSA and MSSA cultures. The antibacterial effects of polyphenols were evaluated using a liquid dilution method. Briefly, pre-cultured bacterial solutions of 104 colony forming units (CFU)/well on 96-well microplates were incubated in the presence of serial two-fold dilutions of the compounds at 32 °C for 24 h. MICs of the compounds were defined as the lowest concentrations at which the bacterial culture lacked visual turbidity after the incubation. The effects of theasinensin A on the MIC values of the antibiotics were estimated by incubation in the presence of theasinensin A at concentrations lower than the MIC value. The effects of gallic acid were examined in an analogous way.

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Effects of the addition of theasinensin A on the cell growth profile of MRSA in the presence of oxacillin

To each solution containing 106 CFU/mL of the MRSA OM584 strain, theasinensin A, oxacillin, or a mixture of the two compounds was added respectively, and the solution was incubated at 32 °C. To construct growth profiles, the amount of bacteria in each solution was determined based on turbidity monitored by light absorbance at 650 nm.

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Estimation of amounts of viable MRSA cells in the tubes during the incubation in the presence/absence of theasinensin A and oxacillin

An aliquot of pre-cultured bacteria at 106 CFU/mL was incubated at 32 °C for 48 h in the presence or absence of theasinensin A and/or oxacillin. An aliquot of this culture solution was then incubated on nutrient agar broth at 37 °C for 24 h to visualize the number of bacterial colonies, which corresponds to the number of viable cells.

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Detection of penicillin-binding protein (PBP) 2′ in the MRSA cells cultured in the presence of theasinensin A or oxacillin

Pre-cultured bacteria were incubated in the presence of theasinensin A or oxacillin until the absorbance at 650 nm reached 0.7 and 200 μL of the cell cultures were then centrifuged at 10,000 rpm for 5 min. After the supernatant had been removed, the residue was washed twice by re-suspension in a 0.05 M phosphate buffer and centrifugation (15,000 rpm for 8 min); the residue was then treated with 1 M NaOH, followed by treatment with 0.5 M KH2PO4. The mixture was centrifuged at 4,500 rpm for 5 min to give a PBP fraction as a supernatant. The presence of PBP2′ in the fraction was detected by coagulation with anti-PBP2′ monoclonal antibody-sensitized latex on a test card (MRSA Screen, Denka Seiken, Japan). The amount of PBP2′ produced was estimated semi-quantitatively using Scion Image software (Scion, Frederick, Maryland, USA).

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Results and Discussion

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Decrease of EGCG in neutral solutions

The incubation of EGCG in phosphate buffer solutions at 37 °C caused structural changes as shown by the HPLC analysis even in the neutral conditions (Fig. [1]). Fig. [2] shows the decrease in the amount of EGCG during the incubation at pH 6.5 - 7.8. Table [1] lists the half-lives (t1/2) of EGCG under the different experimental conditions. The rate of EGCG decay was dependent on its initial concentration. The addition of a chelating reagent (DETAPAC) slowed the oxidation reaction; the subsequent addition of Fe3+ ion restored the half-life value to that observed before the additions of DETAPAC or Fe3+. These data suggested the participation of trace amounts of metal ion(s), such as Fe3+, in the oxidation process. The concentration-dependence of EGCG oxidation could be explained by the chelating properties of the phenolic hydroxy groups on the EGCG molecule.

The products formed from EGCG were isolated and identified as theasinensins A and D, (-)-gallocatechin gallate, and gallic acid. The HPLC analysis showed that the main product was theasinensin A. The amount of theasinensin A was up to 16 %, w/w based on the amount of EGCG, after incubation for 2 h (at pH 7.4), at which time almost all of the EGCG was gone.

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Zoom Image

Fig. 1 HPLC profile of a reaction mixture obtained after incubation of EGCG in a phosphate buffer of pH 7.0 (5.5 × 10 - 4 M) for 3 h. a, gallic acid; b, theasinensin A; c, EGCG; d, theasinensin D; e, (-)-gallocatechin gallate.

Zoom Image

Fig. 2 Decrease of EGCG in phosphate buffers of pH 6.5 - 7.8 at 37 °C. -○- pH 6.5; -•- pH 7.0; -□- pH 7.4; -▴- pH 7.8. EGCG (5.5 × 10 - 4 M) in each of the phosphate buffers (1.0 mL) was kept in a tube at 37 °C in a water-bath and the content of EGCG in the solution was determined by HPLC. The slight increase in pH caused a faster decrease of EGCG.

Table 1 Effects of changes in the conditions on the half-life of EGCG in phosphate buffer solution
Conditions
pH EGCG concentration
(M)		 Additive t1/2 (h)a
7.4 5.5 × 10 - 4 none 0.56
7.4 5.5 × 10 - 4 DETAPAC (2.7 × 10 - 3 M) 0.75
7.4 5.5 × 10 - 4 DETAPAC (2.7 × 10 - 3 M)
+ FeCl3 (4.3 × 10 - 4 M)		 0.52
7.4 2.2 × 10 - 3 none 2.33
7.4 8.7 × 10 - 3 none 8.78
6.5 5.5 × 10 - 4 none 9.39
7.0 5.5 × 10 - 4 none 2.08
7.8 5.5 × 10 - 4 none 0.43
a The values are means from triplicate experiments.
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Antibacterial effects of the polyphenols produced from EGCG

The compounds formed from EGCG were examined for their antibacterial effects against MRSA strains using a microliquid dilution assay. The MIC values of the compounds are shown in Table [2]. The dimeric products, theasinensins A and D, had MIC values slightly higher than that of EGCG, although the values indicated on the molar basis were equal to that of EGCG. Gallic acid displayed weaker antibacterial activity against the MRSA strains.

Table 2 MICs (μg/mL) of EGCG and polyphenols formed from EGCG against four MRSA strains and the effects of the addition of theasinensin A or gallic acid on the MICs (μg/mL) of oxacillin for the MRSA strains
MRSA MSSA
OM481 OM505 OM584 OM623 209P
EGCG 32 32 32 32 32
(7.0)* (7.0) (7.0) (7.0) (7.0)
Theasinensin A 64 64 64 64 64
(7.0) (7.0) (7.0) (7.0) (7.0)
Theasinensin D 64 64 64 64 64
(7.0) (7.0) (7.0) (7.0) (7.0)
Gallic acid 128 128 128 128 128
(75) (75) (75) (75) (75)
Oxacillin 256 64 256 256 < 0.5
Oxacillin + Theasinensin A (1.8 × 10 - 5 M) 128 64 32 128 < 0.5
Oxacillin + Theasinensin A (3.5 × 10 - 5 M) 4 4 4 4 < 0.5
Oxacillin + Gallic acid (2.9 × 10 - 4 M) 2 32 32 64 < 0.5
*The values in parentheses are given in molar concentrations (× 10 - 5 M).
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Effects of theasinensin A on the MIC values of oxacillin against the MRSA strains.

The effects of theasinensin A on the antibiotic resistance of the MRSA strains are shown in Table [2] and Table [3]. The MICs of oxacillin for the MRSA strains were 256 μg/mL (for MRSA OM481, OM584, and OM623 strains) or 64 μg/mL (for MRSA OM505 strain) in the absence of theasinensin A; the MIC for the MSSA 209P control strain was below 0.5 μg/mL. However, upon the addition of theasinensin A [3.5 × 10 - 5 M (32 μg/mL); half of the MIC of theasinensin A], the MICs of oxacillin decreased to 4 μg/mL for all of the MRSA strains. Gallic acid noticeably decreased the MIC of oxacillin for the MRSA OM481 strain; the MIC of oxacillin decreased to 2 μg/mL when 2.9 × 10 - 4 M (50 μg/mL) of gallic acid was added.

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Effects of theasinensin A on the MIC values of other antibiotics against the MRSA strains

Furthermore, the antibiotic resistance of the MRSA strains against β-lactams other than oxacillin was affected in the presence of theasinensin A. In the presence of theasinensin A, the MIC values of penicillin G and ampicillin for the MRSA strains decreased to 1/256 - 1/16 of those in the absence of theasinensin A, as shown in Table [3].

The effects of theasinensin A on the MIC values of other types of antibiotics were also examined. Although the MIC values of erythromycin (a macrolide antibiotic), tetracycline, and fosfomycin for the MRSA strains were not affected by the addition of theasinensin A, that of streptomycin, an aminoglycoside, decreased remarkably, by 1/32 - 1/2, in the presence of theasinensin A (Table [3]). It is notable that the MIC of streptomycin also decreased by 1/32 for the MSSA 209P strain in the presence of theasinensin A (3.5 × 10 - 5 M). These results suggested that in order to suppress the adverse effects of aminoglycoside antibiotics, the required dose of these drugs could be reduced by the addition of theasinensin A.

Table 3 Effects of theasinensin A on the MICs (μg/mL) of β-lactams and other classes of antibiotics for the MRSA strains
MRSA MSSA
OM481 OM505 OM584 OM623 209P
Penicillin G 32 32 32 32 < 0.5
Penicillin G + Theasinensin A (8.8 × 10 - 6 M) 32 32 16 32 < 0.06
Penicillin G + Theasinensin A (1.8 × 10 - 5 M) 4 8 4 4 < 0.06
Penicillin G + Theasinensin A (2.6 × 10 - 5 M) 2 8 2 2 < 0.06
Penicillin G + Theasinensin A (3.5 × 10 - 5 M) 0.125 0.5 0.25 0.25 < 0.06
Ampicillin 16 32 32 32 < 0.5
Ampicillin + Theasinensin A (2.7 × 10 - 5 M) 1 2 1 2 < 0.5
Ampicillin + Theasinensin A (3.5 × 10 - 5 M) 0.5 1 0.5 0.5 < 0.5
Streptomycin 4 8 8 16 4
Streptomycin + Theasinensin A (2.7 × 10 - 5 M) 1 8 4 8 0.5
Streptomycin + Theasinensin A (3.5 × 10 - 5 M) 0.125 4 2 4 0.125
Erythromycin > 128 > 128 > 128 > 128 0.125
Erythromycin + Theasinensin A (3.5 × 10 - 5 M) > 128 > 128 > 128 > 128 < 0.06
Tetracycline 4 0.25 64 64 0.125
Tetracycline + Theasinensin A (3.5 × 10 - 5 M) 1 0.25 64 64 < 0.06
Fosfomycin > 128 > 128 > 128 > 128 4
Fosfomycin + Theasinensin A (3.5 × 10 - 5 M) > 128 > 128 > 128 > 128 2
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Effects of the presence of both theasinensin A and oxacillin on the growth curve of MRSA

The growth curve observed for the MRSA OM584 strain incubated with both oxacillin and theasinensin A revealed a suppression of the turbidity increase, as shown in Fig. [3]. The estimation of viable bacterial cells during the incubation corroborated the bactericidal effect of this treatment combination, as shown in Fig. [4]. The suppressive effect lasted about 10 h, after which another addition of these compounds again suppressed an increase in viable MRSA cells.

The antibiotic resistance of MRSA against β-lactams has been attributed primarily to the production of penicillin binding protein2′ (PBP2′). In order to determine whether the suppression of antibiotic resistance is due to the inhibition of PBP2′ production, the effect of theasinensin A on PBP2′ production was examined. The latex agglutination assay (Fig. [5]) demonstrated that PBP2′ production was not suppressed in the presence of theasinensin A. Alternatively, it is possible that the suppression of antibiotic resistance by theasinensin A may be due to an effect on the ability of PBP2′ to construct the cell wall, or it may be due to direct damage to the bacterial cell surface.

Zoom Image

Fig. 3 Suppressive effect of the combination of theasinensin A and oxacillin shown by the cell growth profile of MRSA OM584 during incubation. -•- control; - oxacillin (5 μg/mL); -▴- theasinensin A (32 μg/mL); -○- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL). Turbidity measured by the absorbance at 650 nm is an indicator of the amount of bacterial cells. The presence of both the antibiotic oxacillin and the polyphenol theasinensin A suppressed the cell growth of MRSA OM584 over 10 h; growth was more weakly suppressed in the absence of either compound.

Zoom Image

Fig. 4 Bactericidal effect of the combination of theasinensin A and oxacillin shown by the change in the viable cell numbers of OM584 during incubation. -✦- control; -□- oxacillin (5 μg/mL); -▵-theasinensin A (32 μg/mL); -✧- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL); -▴- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) and further addition of oxacillin (5 μg/mL) at 10 h after the start of incubation; -○- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) and further addition of theasinensin A (32 μg/mL) at 10 h after the start of incubation; - oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) and further addition of oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) at 10 h after the start of incubation.

Zoom Image

Fig. 5 Effect of theasinensin A and oxacillin on the production of PBP2′ in MRSA OM481. Solutions containing MRSA OM481 were incubated in the presence or absence of theasinensin A or oxacillin. The PBP2′ formed was visualized using the latex agglutination assay, and the amount was estimated semi-quantitatively using the Scion Image software. The strain MSSA 209P, which does not form PBP2′, was used as a negative control. The values are from triplicate experiments, and the bars on the columns indicate standard deviations.

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Conclusion

EGCG rapidly decayed in neutral solutions to give several products, including dimeric compounds such as theasinensins A and D. This process is explained mainly as oxidation in the presence of trace amounts of metal ions, such as Fe3+. Theasinensin A, a major product of the oxidation of EGCG, effectively suppresses the oxacillin resistance of MRSA. The resistance of MRSA to other β-lactams and the aminoglycoside streptomycin was also suppressed by theasinensin A. The presence of both oxacillin and theasinensin A effectively decreased the number of viable MRSA cells, and the effect lasted about 10 h. Repeated administration of both compounds prolonged the effect. The effects of theasinensin A may be caused by an influence on the function of PBP2′ or another mechanism, such as a direct effect on the MRSA membrane. Since EGCG is easily converted into oxidative polyphenolic products under mild conditions, oxidative products, especially theasinensin A, could play important roles in the pharmacological effects reported for tea polyphenols or EGCG. Although parenteral application of tea polyphenols may be required to attain concentrations available for clinical practice because of their low bioavailability [10], their toxicity must be clarified for the use.

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Dr. T. Hatano

Faculty of Pharmaceutical Sciences

Okayama University

Tsushima

Okayama 700-8530

Japan

Fax: +81-86-251-7936

Email: hatano@pharm.okayama-u.ac.jp

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References

  • 1 Okuda T, Mori K, Hatano T. Relationship of the structures of tannins to the binding activities with hemoglobin and methylene blue.  Chemical & Pharmaceutical Bulletin. 1985;  33 1424-33
    PubMedSearch in Google Scholar
  • 2 Okuda T, Kimura Y, Yoshida T, Hatano T, Okuda H, Arichi S. Studies on the activities of tannins and related compounds from medicinal plants and drugs. I. Inhibitory effects on lipid peroxidation in mitochondria and microsomes of liver.  Chemical & Pharmaceutical Bulletin. 1983;  31 1625-31
    PubMedSearch in Google Scholar
  • 3 Iwata S, Fukaya Y, Nakazawa K, Okuda T. Effects of the oxidative damage of mouse ocular lens I. Using the oxidative damage model induced by the xanthine-xanthine oxidase system.  Journal of Ocular Pharmacology. 1987;  3 227-38
    PubMedSearch in Google Scholar
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Dr. T. Hatano

Faculty of Pharmaceutical Sciences

Okayama University

Tsushima

Okayama 700-8530

Japan

Fax: +81-86-251-7936

Email: hatano@pharm.okayama-u.ac.jp

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Fig. 1 HPLC profile of a reaction mixture obtained after incubation of EGCG in a phosphate buffer of pH 7.0 (5.5 × 10 - 4 M) for 3 h. a, gallic acid; b, theasinensin A; c, EGCG; d, theasinensin D; e, (-)-gallocatechin gallate.

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Fig. 2 Decrease of EGCG in phosphate buffers of pH 6.5 - 7.8 at 37 °C. -○- pH 6.5; -•- pH 7.0; -□- pH 7.4; -▴- pH 7.8. EGCG (5.5 × 10 - 4 M) in each of the phosphate buffers (1.0 mL) was kept in a tube at 37 °C in a water-bath and the content of EGCG in the solution was determined by HPLC. The slight increase in pH caused a faster decrease of EGCG.

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Fig. 3 Suppressive effect of the combination of theasinensin A and oxacillin shown by the cell growth profile of MRSA OM584 during incubation. -•- control; - oxacillin (5 μg/mL); -▴- theasinensin A (32 μg/mL); -○- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL). Turbidity measured by the absorbance at 650 nm is an indicator of the amount of bacterial cells. The presence of both the antibiotic oxacillin and the polyphenol theasinensin A suppressed the cell growth of MRSA OM584 over 10 h; growth was more weakly suppressed in the absence of either compound.

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Fig. 4 Bactericidal effect of the combination of theasinensin A and oxacillin shown by the change in the viable cell numbers of OM584 during incubation. -✦- control; -□- oxacillin (5 μg/mL); -▵-theasinensin A (32 μg/mL); -✧- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL); -▴- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) and further addition of oxacillin (5 μg/mL) at 10 h after the start of incubation; -○- oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) and further addition of theasinensin A (32 μg/mL) at 10 h after the start of incubation; - oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) and further addition of oxacillin (5 μg/mL) plus theasinensin A (32 μg/mL) at 10 h after the start of incubation.

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Fig. 5 Effect of theasinensin A and oxacillin on the production of PBP2′ in MRSA OM481. Solutions containing MRSA OM481 were incubated in the presence or absence of theasinensin A or oxacillin. The PBP2′ formed was visualized using the latex agglutination assay, and the amount was estimated semi-quantitatively using the Scion Image software. The strain MSSA 209P, which does not form PBP2′, was used as a negative control. The values are from triplicate experiments, and the bars on the columns indicate standard deviations.