green tea extract weakens the antibacterial effect of amoxicillin in methicillin-resistant...

Green Tea Extract Weakens the Antibacterial Effect of Amoxicillin in Methicillin-resistant Staphylococcus Aureus Infected Mice

Qing Peng,1 Yuanchun Huang,2 Bing Hou,3 Dexing Hua,1 Fen Yao1 and Yuanshu Qian1

1Pharmacology Department, Shantou University Medical College, Shantou, Guangdong, 515041, China2Clinical Laboratory of First Affi liated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China3Clinical Laboratory of Dermatosis Hospital, Shantou, Guangdong, 515041, China

Tea (Camellia sinensis) has been known for its modulation of resistance of methicillin-resistant Staphylococcus aureus (MRSA) to b-lactam antibiotics in vitro. This study aimed to confi rm the in vitro effect of green tea extracts with b-lactams and to determine whether green tea extracts can reduce the minimum inhibitory concentrations (MICs) of amoxicillin in MRSA-infected mice. The catechins in the test tea that account for the reduced resistance to b-lactams were quantitatively determined by high-performance liquid chromatogra-phy. The MICs of the ampicillin, cefazolin, amoxicillin, oxacillin, tea extract alone and tea extract in combina-tion with b-lactams were determined. Proportions of tea extracts and amoxicillin-tea extract combinations were administered to groups of mice enterally. The in vitro experiment showed that the MICs of four b-lactams were greatly decreased in the presence of 0.25% tea extract. However, in an in vivo experiment, amoxicillin in combination with 5% tea extract conferred a higher ED50 than that of antibiotic alone. Green tea extract, alone or in combination with amoxicillin, does not have protective benefi ts in MRSA-infected mice. This study concluded that tea extract weakened the antibacterial effect of amoxicillin in MRSA infected mice. Tea drinking is not recommended in combination with amoxicillin treatment. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: tea; β-lactams; catechins; MRSA; amoxicillin.

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) accounts for a large proportion of hospital-acquired infections and is considered a serious problem because of its multi-drug-resistant properties (Fishbain et al., 2003; Kuehnert et al., 2005). Currently, vancomycin and its analog teicoplanin are the most effective antibiotics for MRSA infection. However, their clinical use often results in unexpected side effects and the development of vancomycin-resistant S. aureus infection (Bailie and Neal, 1988; Berger-Bachi et al., 1992). The search for better drugs to combat this infection is urgently needed.

About a decade ago, much attention was focused on the exploration and utilization of plant extracts (phyto-chemicals) as alternatives to or synergistic enhancers of antibiotics used to treat MRSA infection (Iinuma et al., 1994; Lee et al., 2007; Liu, 2000; Yu et al., 2005). The MICs of some β-lactams used in an in vitro experiment against MRSA were reduced when used in combination with extracts of Japanese green tea (Camellia sinensis) (Yamazaki, 1996). Other studies have investigated the pharmacological potential of green tea. Green tea con-tains high concentrations of catechins and its derivatives

(Horie and Kohata, 1998). Two catechins, (−)-epigal-locatechingallate (EGCg) and (−)-epicatechingallate (ECg) are the components that account for reduced resistance to β-lactams caused by green tea. The other two catechins in green tea, (−)-epigallocatechin (EGC) and (−)-epicatechin (EC), cannot modulate β-lactam resistance but act synergistically with ECg and EGCg.

However, research of in vivo models of β-lactams combined with green tea extracts is lacking. The study aimed to confi rm the in vitro effects of green tea extract combined with β-lactams and to determine whether green tea extracts would enhance the effect of β-lactam when used to treat mice infected with MRSA.

MATERIALS AND METHODS

Tea extract. Aqueous tea extract was prepared with Chinese green tea (Longjin) by adding 100 mL boiling water to cylinders containing 5 g dried tea leaves. After 30 min of aqueous extraction, the tea extract was fi l-tered through a 0.22 μm fi lter. The 5% nominal tea extract concentration was therefore obtained.

High-performance liquid chromatography. ECg, EGCg, EGC and EC in the tested tea were quantitatively determined as described by Khokhar et al. (1997). EC, EGC, EGCg and ECg used for calibration were purchased from Shanghai Usea Biotech Company

* Correspondence to: Y. S. Qian, Pharmacology Department, Shantou University Medical College, Shantou, Guangdong, 515041, China.E-mail: [email protected]

Received 11 February 2009Revised 26 May 2009

Copyright © 2009 John Wiley & Sons, Ltd. Accepted 29 May 2009

PHYTOTHERAPY RESEARCHPhytother. Res. 24: 141–145 (2010)Published online 3 August 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2952

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 141–145 (2010)DOI: 10.1002/ptr

142 Q. PENG ET AL.

(Shanghai, China). The 5% nominal extract was diluted 5 times with water for HPLC samples.

Antibiotics. All antibiotics, including ampicillin, cefazo-lin amoxicillin and oxacillin, were purchased from Sigma Chemical Co. (St Louis, USA).

Culture media. Cation-adjusted Muller-Hinton broth (CAMHB) and Muller-Hinton agar manufactured by Oxoid Ltd (Basingstoke, Hampshire, England) were used.

Bacterial strains. The 15 MRSA isolates used in this study were obtained from the Clinical Microbiology Laboratory of the First Affi liated Hospital of Shantou University, Shantou, China. The selected strains were defi ned as resistant on the basis of sensitivity to oxacillin by the broth tube dilution method in accordance with Clinical Laboratory and Standards Institute (CLSI) guidelines (2007). The standard strain of S. aureus (ATCC 25923) which is a MSSA (methicillin-sensitive Staphylococcus aureus) was used for a control strain.

Animals. Kunming mice (Mus musculus cataneus) weighing 18–22 g were used in this study. Investigation conformed to the Guide for the Care and Use of Labora-tory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All experimental protocols were approved by the Labora-tory Animal Ethics Committee of our institution.

Minimum inhibition concentration determination. The standard agar dilution method was used to determine the MIC of the antibiotics alone in accordance with CLSI (Clinical and Laboratory Standards Institute)guidelines (2007). Ampicillin, cefazolin, amoxicillin and oxacillin were serial diluted to determine the MICs of antibiotics. To determine the MIC of tea extract, the tea extract was diluted by water to give a range of concen-trations from 0.325% to 5% with a volume of 2 mL. 8 mL agar was then mixed with the above serial dilu-tions of tea extract to give a total volume of 10 mL in each plate. Therefore, the fi nal concentrations of tea extract were from 0.0625% to 1% in each agar plate. A fi nal inoculum of 104 CFU (colony-forming units)/mL of 15 MRSA isolates and standard strain (ATCC 25923) were spotted onto the agar plates. The plates were then incubated at 35 ºC for 18 h. The MICs were defi ned as the lowest concentration at which no visible growth was observed.

Checkerboard micro-titer tests. The antibacterial effects of a combination of tea extract and four antimicrobial agents were assessed by checkerboard micro-titer tests (Yu et al., 2005). The antimicrobial combinations assayed included 0.25% tea extract plus serial dilutions of ampicillin, cefazolin, amoxicillin and oxacillin. Serial dilutions of antibiotics and tea extract were mixed in CAMHB. Inocula were prepared from colonies grown on Mueller-Hinton agar after overnight culture. The fi nal bacterial concentration after inocula-tion was 104 colony-forming units (CFUs). After 18 h incubation at 35 ºC, the MIC was determined to be the lowest concentration that completely inhibited the growth of the bacteria.

Toxicity analysis of tea extract via gastric perfusion. A random sample of 40 mice was selected. The mice were divided into two groups for treatment: 0.5% tea extract and 5% tea extract via gastric perfusion (0.5 mL/20 g). Each group had an equal number of males and females. The animals were observed for 14 days for signs of acute toxicity.

Analysis of toxicity with amoxicillin alone and in com-bination with 0.5% and 5% tea extract. A random sample of 15 mice, regardless of sex were divided into three groups (fi ve mice each) for gastric perfusion of amoxicillin (0.5 mL/20 g): 100 mg/kg amoxi cillin diluted in 0.5% carboxymethylcellulose, 100 mg/kg amoxicillin dissolved in 0.5% tea extract, and 100 mg/kg amoxicillin dissolved in 5% tea extract. The animals were observed for 14 days for acute signs of toxicity.

Determination of the minimum lethal dose of bacteria in mice. MRSA Strain No.8 was used to determine the minimum lethal dose (MLD) of the bacteria on the basis of its virulence in mice. The inoculum was pre-pared by harvesting and washing bacterial cells that were cultured in CAMHB at 35 ºC for 18 h. Phosphate buffered saline (PBS) was used to wash the bacterial cells. After washing three times, the MRSA bacterial cells were suspended in sterile PBS that contained 5% gastric mucin. Five dilutions of bacteria, ranging from 105 to 109 CFU/mL, were inoculated in random samples of mice (fi ve mice each), without any preference to sex. Each mouse was intraperitoneally injected with 0.5 mL of MRSA inoculate. The MLD of bacteria causing 100% mortality in a group of mice was recorded 7 days after inoculation.

In vivo effi cacy of amoxicillin combined with tea extract in mice systemic infection model. Mice were intraperitoneally injected with 0.5 mL of MLD inoculum.

Control. A random sample of 10 males and 10 females was selected for the control group, which received no intervention. 1 h after bacterium inoculation, the mice were randomly assigned to receive the following treat-ment interventions.

Amoxicillin. Doses of 6.25, 12.5, 25, 50 and 100 mg/kg were administered via gastric perfusion twice, with an interval of 4 h, to fi ve groups of mice (10 males and 10 females per group).

Amoxicillin plus tea extract. The fi ve doses of amoxicil-lin were added to solutions containing 0.5% and 5% of tea extract. Each combination was tested in fi ve groups of mice (10 males and 10 females per group) twice via gastric perfusion(0.5 mL/20 g), with an interval of 4 h.

Tea extract. Two groups of mice (10 males and 10 females per group) received 0.5% and 5% of tea extract (0.5 mL/20 g) via gastric perfusion.

The treatment groups were observed for 7 days. The mean effective dose (ED50) suffi cient to protect mice and 95% confi dence limits were calculated by the Bliss method (Bliss, 1938).

GREEN TEA EXTRACT ON AMOXICILLIN EFFECT 143


RESULTS

EGC, ECg, EGCg and EC quantitative determination in Chinese green tea

The contents of EGC, EGCg and EC were 77, 525 and 52 μg/mL, respectively, but ECg could not be detected (limit of detection 10 μg/mL) in the 1% tea extract.

MICs of b lactams alone and in combination with tea extract

The results of the MIC tests are shown in Table 1. The minimum concentration of tea extract that could inhibit the growth of 15 MRSA isolates and MSSA (ATCC 25923) in culture medium was 5 mg/mL (0.5%). A 0.25% concentration of green tea extract (1/2 MIC of tea alone) was able to markedly reduce the MIC of ampicillin, cefazolin, amoxicillin and oxacillin against MRSA and MSSA (ATCC 25923).

Acute toxicity experiment

No abnormal symptoms were observed in all tested mice after oral administration of tea extracts, amoxicil-lin or amoxicillin–tea extract combined.

In vivo effects of tea extract alone and in combination with amoxicillin

The ED50 for amoxicillin combined with 0.5% tea extract treatment was similar to that of amoxicillin alone. However, the ED50 for amoxicillin combined with 5% tea extract was two-fold higher than that for amoxicillin alone (Table 2).

DISCUSSION

The continuing research on catechins has shed more light on the promising ability of green tea extracts to

Table 1. Minimal inhibitory concentrations (MICs) of b-lactams alone and in combination with 0.25% green tea extract against 15 MRSA isolates and MSSA (ATCC 25923)

Strain

MIC (μg/mL)

AMP AMP + tea CEZ CEZ + tea AMO AMO + tea OXA OXA + tea

MRSA 1 128 1 128 1 256 2 128 2MRSA 2 64 0.5 64 0.25 256 32 128 1MRSA 3 128 2 256 4 256 0.25 256 4MRSA 4 128 2 128 1 256 4 256 8MRSA 5 64 32 8 0.125 64 0.25 64 1MRSA 6 256 2 64 0.5 256 2 128 16MRSA 7 128 2 64 1 256 2 128 8MRSA 8 128 0.5 128 0.5 64 0.25 128 2MRSA 9 128 2 128 0.25 128 16 256 1MRSA 10 128 1 128 32 256 0.25 128 0.5MRSA 11 16 0.125 128 2 32 0.25 256 32MRSA 12 128 0.25 64 0.25 256 4 256 16MRSA 13 128 0.25 128 0.5 256 2 256 2MRSA 14 128 0.5 128 16 256 64 256 4MRSA 15 128 2 128 0.5 256 0.25 256 16ATCC25923 0.25 0.0625 1 0.25 0.5 0.125 2 0.25

AMP, ampicillin; CEZ, cefazolin; AMO, amoxicillin ; OXA, oxacillin.

Table 2. Effect of green tea extract in combination with amoxicillin by oral administration

Drug Dose (mg/kg) No. of mice No. of deaths ED50 (95% confi dence limit) (mg/kg)

Amoxicillin 100 20 050 20 425 20 612.5 20 12 17.399 (13.236–22.872)6.25 20 18

Amoxicillin + 0.5% tea extract 100 20 050 20 325 20 712.5 20 14 17.852 (13.653–23.343)

6.25 20 17Amoxicillin + 5% tea extract 100 20 4

50 20 825 20 1412.5 20 18 42.264 (31.849–56.085)6.25 20 20

0.5% tea extract – 20 205% tea extract – 20 20Infected control group – 20 20


144 Q. PENG ET AL.

help combat infections. EGCg and ECg are two cate-chins in Japanese green tea extracts that have antibacte-rial effects (Stapleton et al., 2004). Some evidence shows that EGCg binding directly to the peptidoglycan layer interferes with the synthesis of the cell wall, ultimately damaging the protective layer of bacteria (Shimamura et al., 2007). However, ECg inserts into the cytoplasmic membrane, releases lipoteichoic acids and eventually effects changes in the structure of the teichoic acid of the cell wall (Stapleton et al., 2007). This situation, in turn, may make it easy for β-lactams to target the cell wall of bacteria. Nongalloylated catechins such as EC and EGC may increase the ECg membrane binding and can enhance the oxacillin susceptibility of MRSA strains by the galloyl catechins ECg and EGCg (Stapleton et al., 2006). The above mechanism could be the under-lying reason for the fi nding that green tea extract enhances the effects of β-lactams in in vitro experi-ments. A reduction of the MICs of β-lactams against MRSA in the presence of 0.25% tea extract was observed. On HPLC, EGCg was the main active com-ponent in the tested Chinese tea that contributed to the enhancing effect of β-lactams against MRSA. However, ECg was not detectable on HPLC. This variation in the content of green tea from different places may due to the species of plant, soil and atmospheric conditions. Considering the lack of detectable ECg in the test green tea extract, further experiments are required to investi-gate the effects of pure ECg in combination with β- lactams in vitro and in vivo.

Our in vivo testing of mice models did not demon-strate a synergism between green tea extracts and β-lactams against MRSA. Combinations of amoxicil-lin–tea extract could not control the induced infection in the tested animals. Furthermore, increasing the con-centration of green tea extract in amoxicillin–tea extract combination led to only a reduction of its effi cacy. One factor counteracting the results observed in in vitro studies should be the poor bioavailability of EGCg. EGCg has a large number of hydrogen bond donors (i.e. hydroxyl groups), has a large polar molecular surface area and is predicted by ‘Lipinski’s Rule of 5’ to be

poorly absorbed (Clark, 1999; Lipinski et al., 2001). Moreover, EGCg is subjected to extensive glucuronida-tion, methylation and sulfation, as well as microbial degradation in the colon (Kida et al., 2000; Lu et al., 2003a, 2003b). Gastrointestinal tract features such as limited membrane permeability, transporter-mediated intestinal secretion, or gut wall metabolism may con-tribute signifi cantly to the low bioavailability of cate-chins from green tea taken orally (Cai et al., 2002). The poor bioavailability of EGCg may also be due to the susceptibility of catechins to hydrolysis by intestinal bacterial and mammalian esterases (Kohri et al., 2001). In addition, proteins can interact with catechins and form protein precipitates (Sekiya et al., 1984; Shimamura et al., 2007). Serum proteins and other pro-tein-based biomolecules in living systems could act in the same way and affect the bioavailability and subse-quent effects of catechins. It was reported that the rate of ampicillin and amoxicillin bioavailability were reduced signifi cantly by khat chewing for the reason that β-lactams in general are nitrogenous compounds that could combine with catechins in khat, leading to the formation of insoluble products that can affect the antibacterial capacity and bioavailability of β-lactams (Attef et al., 1997). This situation could also explain why the amoxicillin–5% tea extract combination had higher ED50s than antibiotic alone in our in vivo experiment.

To improve the absorbability of catechin gallates or enhance their anti-MRSA effects, some researchers have attempted to modify the chemical structures of EGCg and ECG (Lambert et al., 2006; Yajima et al., 2007), which might hold the key to their proper utilization.

In conclusion, this study has added support to green tea extract possibly reversing the resistance of MRSA to β-lactams in vitro, but the low bioavailability of cat-echins in green tea limits its use in treating MRSA infection. Furthermore, our fi ndings indicate that the antibacterial effect of amoxicillin is weakened by oral administration of tea extracts in MRSA infected mice. Therefore, tea drinking is not recommended in combi-nation with amoxicillin treatment.

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