Int J Pharm Pharm Sci, Vol 7, Issue 6, 258-262Original Article


ANTIBACTERIAL ACTIVITY OF GREEN TEA EXTRACT IN COMBINATION WITH CEFOTAXIME ON DIARRHEA CAUSING ESBL PRODUCING E. COLI

RATHOD SEJAL*, WILLIAMSON MANITA

Department of Microbiology, Topiwala National Medical College and B. Y. L. Nair charitable Hospital, Mumbai 400008, India
Email: sejjit@yahoo.com

Received: 26 Mar 2015 Revised and Accepted: 28 Apr 2015

ABSTRACT

Objective: Owing to the continuous evolution of antibiotic resistance, treatment with natural products represents significant adjuncts or alternatives to conventional antibiotic therapy. Enterobacteriaceae especially Escherichia coli have acquired antibiotic resistance due to production of Extended spectrum Beta-lactamases (ESBL).

Methods: Effect of Aqueous extract of Green tea (AGTE) was studied on 44 ESBL producing, diarrhea causing E. coli. ESBL production of the strains was confirmed by phenotypic confirmatory disc diffusion test (PCDDT) and E-test. Antibacterial activity of AGTE was studied by disc diffusion method and the Minimum Inhibitory Concentration (MIC) of AGTE and Cefotaxime were determined by an agar dilution technique. The combined activity of AGTE and Cefotaxime was determined by calculating the fractional inhibitory concentration (FIC index) by the checkerboard method. The active ingredients epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG) present in the extract were quantitated by High Performance liquid Chromatography (HPLC).

Results: AGTE exhibited significant antibacterial activity as the observed zones of inhibition ranged from 10–26 mm against the 44 ESBL producing E. coli strains. The MIC of AGTE was found to be in the range of 2.5-20 mg/ml with a mean of 7.23 mg/ml. 8 test strains showed synergism whereas the remaining showed an additive effect between AGTE and Cefotaxime. By HPLC analysis the concentration of EC, ECG, EGC, EGCG were found to be 7.03, 10.08, 9.99 and 43.57 respectively, expressed as percentage w/w.

Conclusion: As green tea extract has shown substantial antibacterial as well as synergistic activity, it can be of potential therapeutic value against ESBL producing Escherichia coli gastrointestinal infections.

Keywords: ESBL, Green tea, EGCG, Synergy.

INTRODUCTION

Drug resistance is a natural step in microbial evolution, with many microorganisms, especially of the Enterobacteriaceae family being highly adaptable and easily capable of mutating to acquire means of exhibiting drug resistance. Since β-Lactam antibiotics are the most widely used, it is therefore not surprising that resistance to many β-lactams is common and still evolving. Mechanisms of resistance to β-lactams in Escherichia coli can be divided into three categories, namely, enzymatic inactivation of the antibiotic, alteration of the target, and reduced penetration of the antibiotic. Enzymatic inactivation to β-lactams is most often caused by the production of enzymes extended spectrum β-lactamases (ESBLs).

Over the last 15 years, numerous outbreaks of infection with organisms producing ESBLs have been observed worldwide [1]. The advent of ESBLs producers has posed a great threat to the use of many classes of antibiotics particularly cephalosporins. There are indications that poor outcome occurs when patients with serious infections due to β-lactamase producing organisms are treated with antibiotics to which the organism is resistant [2].

Extended spectrum Beta-lactamases (ESBL) hydrolyze extended spectrum cephalosporins and are inhibited by Clavulanic acid, Sulbactum and Tazobactum [3]. E. coli has been revealed to be one of the most commonly encountered ESBL producing organisms in various parts of India [4-6]. Treatment of such infections with a combination of a β-lactam with a β-lactamase inhibitor is common, but it may not always prevent the emergence of resistance [7].

Several studies in the recent past emphasized the urgent need for new therapeutic strategies, including the use of plant extracts and phytochemicals extracted from them [8,9]. Such an approach can help curb or considerably decrease the occurrence of drug resistance among bacteria as plant extracts usually have multiple active ingredients that act at various target sites in the bacteria.

Green tea (Camellia sinensis leaves) has been consumed since ancient times in China and has gained popularity in several other countries including India. Green tea extracts being a popular beverage consumed worldwide, inexpensive, and non-toxic, serves as a very important a source of alternative medicine. It is known to possess preventive activity against cardiovascular diseases and some forms of cancer [10]. Also, it has antioxidant [11], antitumor, anti-inflammatory, anti diabetic, immunomodulatory [12] and antibacterial activities [8,9]. Green tea consists of catechins viz. epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG). Other polyphenols include flavanols and their glycosides [13]. EGCG, a main constituent of green tea polyphenol, has been reported to have great antimicrobial activity and also exhibits synergistic properties with other antibiotics against Gram-positive and Gram-negative bacteria [14-16]. By means of molecular dynamic simulations it was revealed that a number of tea catechins have a strong affinity for the lipid bilayer via hydrogen bonding to the lipid bilayer and among them EGCG showed the strongest interaction as it can form large number of hydrogen bonds.

However the effect of Green tea for diarrhea causing ESBL producing E. coli still needs to be elucidated. The objective of the present study is thus to prepare an aqueous extract of Green tea and validate its antibacterial activity against diarrhea causing ESBL producing E. coli.

MATERIALS AND METHODS

The present study was carried out in the Department of Microbiology, Topiwala National Medical College and BYL Nair Charitable Hospital, Mumbai, India and approved by the local Ethics Committee of our institution.

Bacterial strains

E. coli strains were isolated and identified using standard methods from stool samples obtained from patients suffering with acute diarrhea admitted to our tertiary care hospital.

Determination of antimicrobial susceptibility

Antimicrobial Susceptibility Testing (AST) was performed using the disk diffusion method as described by the Clinical and Laboratory Standard Institute (CLSI) using Kirby-Bauer method [18]. Antibiotic discs were obtained from Himedia Ltd. Antibiotics used were Chloramphenicol (C), Ciprofloxacin (Ci), Nalidixic acid (NA), Amoxyclav (AMC), Ceftriaxone (CTR), Ceftazidime (CAZ), Cefotaxime (CTX), Co-trimoxazole (CO) and Imipenem (I).

Screening for ESBL production

All the isolates showing resistance to 3rdgeneration cephalosporins, namely Ceftazdime, Ceftriaxone and Cefotaxime, were tested for confirmation of β-lactamase production by phenotypic confirmatory disc diffusion test (PCDDT). The Optical Density (O. D.) of the cultures were adjusted to 0.5 McFarland (∼1.5 × 108 CFUmL−1) and swabbed on Mueller-Hinton (MH) Agar plates. The screening was done as per CLSI guidelines [18].

Confirmation of ESBL production by E-test

Confirmation of ESBL production was done using Multi-Ezy MICTM Strips (Hi-Media Laboratories Pvt. Ltd.). These strips differ from the conventional E-strips in that they contain a gradient of 3 antibiotics with and without Clavulanic acid on either side of the strip respectively instead of one antibiotic. The Multi-Ezy MICTM Strips contain Cefotaxime, Ceftazidime and Cefipime (MIX) on one side in a twofold gradient and the same antibiotics with Clavulanic acid (MIX+) on the other side. A ratio of inhibition zones for MIX and MIX+of ≥ 8 was considered as a positive E-test [19]. E. coli ATCC 25922 as negative control and Klebsiella pneumonia ATCC 700603 as positive control were used in the test.

Preparation of green tea extract

The dried Green tea powder was provided by Konark Herbals and Health Care, Mumbai. 10 g of dried powder was boiled with 100 ml of distilled water for 10 min. The heated solution was filtered and evaporated at 40˚C. The aqueous green tea extract (AGTE) thus prepared was then kept in the refrigerator for further use.

High-performance liquid chromatography analysis

The High-performance liquid chromatography (HPLC) system consisted of a Shimadzu LC-2010CHT model (Shimadzu, Tokyo, Japan), with a column of Phenomenex Luna-C18 (4.6 × 250 mm, 5 μm Merck) and detection at a wavelength of 280 nm. Elution was carried out at a flow rate of 1.6 ml/min under a linear gradient of Buffer made up of 0.136 g of anhydrous potassium dihydrogen phosphate in water and 0.5 ml of orthophosphoric acid (solvent A) and acetonitrile (solvent B), with a run time of 35 min. The AGTE was dissolved in water and 20 μl was injected into the HPLC. The presence of epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG) was confirmed by the same retention time of their standards (Natural Remedies, India). The amount of catechins in the sample was calculated using the formula:

Antibacterial activity of AGTE

The antibacterial activity of the extract was tested using the disk diffusion method described by the Clinical and Laboratory Standards Institute. Briefly, sterile paper discs (6 mm; Hi-Media, India) were loaded with 20 μl of extracts at concentration of 500 mg/ml dissolved in sterile distilled water and then left to dry for 18 h at 37◦C. The bacterial suspensions were then diluted to a turbidity of approximately 0.5 McFarland (∼1.5 × 108 CFUmL−1). Ceftazidime/Clavulanic acid (30/10 µg) CAC and Imipenem (10 µg) (Hi-Media, India) was used as the positive control. After incubation at 37◦C for 24 h, the diameter of the zone of inhibition around each of the discs was measured and recorded. Each experiment was performed in triplicate.

Determination of MIC

The MIC value of the AGTE was determined using the Agar dilution technique [19]. The suspensions of all 44 ESBL producing E. coli cultures were adjusted to a turbidity of 0.5 McFarland. The AGTE was added to Mueller and Hinton agar in order to obtain concentrations from 0.05 % to 2 % (0.5 mg/ml to 20 mg/ml) and the plates were poured. Similarly plates containing Cefotaxime in the range of 5 to 1000μg/ml were prepared. Saline suspensions of the E. coli strains were inoculated onto the plates. The lowest concentration capable of inhibiting visible growth after 24 h of incubation at 37◦C was then recorded as the MIC.

Determination of combined activity by Checkerboard assay

Cefotaxime stock (10 mg/ml) and 10 % AGTE was used for the Checkerboard assay. The concentration range of Cefotaxime was from 5 to 1000 μg/ml and AGTE ranged from 0.05% to 2% (0.5 mg/ml to 20 mg/ml). Visible growth after 24 h of incubation at 37◦C was checked and the MIC in combination for Cefotaxime and AGTE were determined for each strain of E. coli. The fractional inhibitory concentration was calculated as follows:

FIC of compound a (FICa) = MIC of compound A (Cefotaxime) in combination/MIC of compound A alone

FIC of compound b (FICb) = MIC of compound B (AGTE) in combination/MIC of compound B alone

The sum of fractional inhibitory concentration (FICs) indices of two compounds in the combination was calculated as follows: FICa+FICb = FICs

FIC indices were interpreted as synergic when values were ≤ 0.5 and as antagonistic when values were>4. The results between synergy and antagonistic tendency (>0.5 and<4) were defined as additive or indifferent. [20]

RESULTS

E. coli strains isolated from stool samples obtained from patients suffering from diarrhea were screened for their susceptibility to antibiotics Chloramphenicol (C), Ciprofloxacin (Ci), Nalidixic acid (NA), Amoxyclav (AMC), Ceftriaxone (CTR), Ceftazidime (CAZ), Cefotaxime(CTX), Co-trimoxazole (CO) and Imipenem (I). The susceptibility pattern of the test strains is as shown in table 1. All the strains were resistant to Ampicillin, Ceftriaxone, Ceftazidime, Cefotaxime and Nalidixic acid. 57% of the strains exhibited resistance towards Amoxyclav, 66% each towards Co-trimoxazole and Ciprofloxacin. 61% were resistant to Chloramphenicol whereas all were sensitive to Imipenem as observed in table 1. Resistance to all the third generation Cephalosporins viz. Ceftriaxone, Ceftazidime, Cefotaxime indicates that the stains are ESBL producers, which was confirmed by PCDDT and E-test. The Phenotypic confirmatory disc diffusion test was positive for all the E. coli strains as an increase in the zone diameter for Ceftazidime-Clavulanic acid by ≥ 5 mm as compared to Ceftazidime alone was observed. E-test using Ezy-MICTM strips confirmed the ESBL production of all the 44 E. coli strains as a ratio of inhibition zones for MIX and MIX+of ≥ 8 was observed. To estimate the amounts of active ingredients EC, ECG, EGC, and EGCG, HPLC analysis was carried out and were found to be 7.03, 10.08, 9.99 and 43.57 respectively, expressed as percentage w/w. EGCG being the main catechin is present in maximum amount in AGTE.

The antimicrobial effect of AGTE was determined using the disc diffusion method, carried out in triplicates. The average zone sizes ranged from 10–26 mm with a mean of 16 mm for the test strains. The individual zone sizes of the test strains are as shown in fig. 1. The MIC of AGTE was determined by the agar dilution technique. Concentrations from 0.05% to 2% (0.5 mg/ml to 20 mg/ml) were used to determine the MIC. MIC of AGTE for the 44 strains ranged from 2.5-20 mg/ml with a mean of 7.23 mg/ml as depicted in table 2. As the organisms are all ESBL producers their MIC values for Cefotaxime were very high. A MIC of ≥ 4 µg/ml indicates ESBL production. As seen in table 3 all the 44 strains showed MIC of Cefotaxime ≥ 10 µg/ml hence clearly highlights the ESBL production of the test strains.

Table 1: Pattern of Antibiotic susceptibility of the E. coli strains

Antibiotic  A AMC C Co Ci NA CAZ CTX CTR I
 No. of strains resistant 44 25 27 29 29 44 44 44 44 0
Percentage of resistant strains 100 57 61 66 66 100 100 100 100 0

Note: n = 44

Table 2: MIC of AGTE against ESBL producing E. coli. Table shows number and percentage of strains inhibited by various concentrations of AGTE

Concentration of AGTE (mg/ml) 0.5 1.0 2.5 5.0 10 15 20 25
No. of strains inhibited - - 13 8 21 - 2 -
Percentage of strains inhibited - - 29.5 18.2 47.7 - 4.5 -

Note: n = 44

Table 3: MIC of Cefotaxime against ESBL producing E. coli. Table shows number and percentage of strains inhibited by various concentrations of Cefotaxime

Concentration of Cefotaxime (µg/ml) 10 50 100 150 200 250 300 350 500 1000
No. of strains inhibited 4 4 7 6 3 2 9 3 - 6
Percentage of strains inhibited 9.1 9.1 15.9 13.6 6.8 4.5 20.5 6.8 0 13.6

Note: n = 44

The combined activity of Cefotaxime and AGTE was determined by checkerboard assay. Calculations of FICs were performed as shown below for strain E8 as an example as shown in fig. 2. For strain E8, FIC a = 50/350 = 0.14. FIC b = 2.5/10 =0.25

So FICs = 0.14+0.25 = 0.39 rounded up to 0.4, which indicates a synergistic effect between Cefotaxime and AGTE. The results obtained for all the 44 strains are as mentioned in table 4 where 8 strains are showing synergistic activity with FICs ≤ 0.5, whereas all the remaining strains showed an additive effect with FICs between 0.5 and 4. None of the strains exhibited Antagonism between AGTE and Cefotaxime.

Fig. 1: Bar chart showing mean zone sizes of AGTE by disc diffusion method. Vertical bars indicate standard deviation (n=3)


Fig. 2: Interpretation of combined effect of AGTE and Cefotaxime by Checkerboard assay for strain E8

DISCUSSION

For the last several years, frequent occurrence of infection with organisms producing β-lactamases has been observed globally [21]. The advent of β-lactamases producers has established a great hazard to the use of many classes of antibiotics particularly cephalosporins. There are indications that poor outcome arises when patients with severe infections due to β-lactamase producing organisms are treated with antibiotics to which the organism is resistant [2]. The high incidence of gastrointestinal tract infections in the general population, the potential for complications, especially in developing countries like India and the associated costs of treatments calls attention to the importance of alternate therapy. In this regard plant extracts are being comprehensively researched by scientists [15, 22, 23].

E. coli strains were isolated from patients suffering from severe diarrhea. Resistance to antimicrobial agents in these species has become an increasingly relevant problem for health care providers. These isolates were confirmed to be ESBL producers by PCDDT and E test. Due to ESBL production these organisms were resistant to third generation cephalosporins. Their resistance was also extended to other groups of antibiotics like Ciprofloxacin and Co-trimoxazole. Many times ESBL producing organisms posses or have acquired genes that encode co-resistance to two or more groups of antibiotics. Management and treatment of infections caused by such resistance pathogens offer a great difficulty and increase health care cost. Therefore, there is a need to investigate alternate methods of therapy.

Green tea is consumed worldwide now and its beneficial physiological and pharmacological effects are very well known. A study by Nima et al. [24] stated the MIC of aqueous green tea for 18 E. coli isolates to be in the range of 75 to 150 mg/ml with a mean of 122 mg/ml. In another study by Tiwari et al. [16] the MIC of green tea extract against E. coli was found to be 88.30 mg/ml. In this study the MIC values ranged from 2.5-20 mg/ml with a mean of 7.23 mg/ml which is much lesser than the previous study suggesting better extraction of tea catechins. There are various studies that have carried out in vivo experiments to demonstrate the significance of Green tea. One such study by Toda et al. [25] proved that a mixture of tea catechins protected rabbits from an experimental infection caused by V. cholerae and suggested that patients with cholera could benefit if tea extracts were added to oral rehydration solutions.

Antibiotic therapy combined with phytochemicals may delay the emergency of bacterial resistance and may also produce desirable synergistic effects in the treatment of infections caused by ESBL producing organisms. This could be due to the fact that crude plant extracts have many different phytochemicals, which inhibit bacteria by different unknown mechanisms. This dual attack of both phytochemicals and antibiotic on different target sites of the bacteria could lead to either an additive or a synergistic effect. AGTE showed a decrease in the MIC of Cefotaxime for all the test strains. 8 strains showed synergism whereas the remaining test strains showed an additive effect. None of the test strains showed antagonistic effect between AGTE and Cefotaxime. Such synergistic activity was also demonstrated by Cui Y. et al. in 2012 [15], Tiwari et al. in 2004 [16] and Zhi-Qing Hu et al. in 2002 [20] on different test organisms. Hence there is no doubt about Green tea being a boon, antimicrobial benefit of which should be reaped in the form of providing lead molecules in developing better and effective drugs by pharmacists.

Table 4: FIC values and results of combined effect of Cefotaxime and AGTE against ESBL producing E. coli

Strain No. FICs Effect Strain no FICs Effect
E1 2.9 A E23 0.6 A
E2 0.1 S E24 0.6 A
E3 1.3 S E25 1.5 A
E4 2.0 A E26 2.3 A
E5 0.8 A E27 2.1 A
E6 0.8 A E28 2.8 A
E7 2.5 A E29 1.6 A
E8 0.4 S E30 2.5 A
E9 0.9 A E31 0.2 S
E10 2.0 A E32 0.3 S
E11 2.1 A E33 2.0 A
E12 2.5 A E34 1.7 A
E13 0.3 S E35 1.3 A
E14 1.5 A E36 0.8 A
E15 1.5 A E37 1.7 A
E16 1.8 A E38 0.8 A
E17 0.4 S E39 1.7 A
E18 1.0 A E40 1.0 S
E19 1.3 A E41 0.6 A
E20 1.5 A E42 0.8 A
E21 0.6 A E43 2.5 A
E22 1.7 A E44 1.6 A

Note: S means Synergistic effect as FICs ≤ 0.5, A means Additive effect with FICs between 0.5 and 4

CONCLUSION

Green tea (Camellia sinensis) extract has been proved to be effective against ESBL producing E. coli not only alone but also in combination with antibiotic. The tea catechins can be purified and tested further in vivo which will help to confirm the therapeutic value of Green tea. Thus, Green tea can be used as an alternate therapy or can be used as an addition to the existing conventional therapy to cure diarrheagenic infections caused by ESBL producing E. coli.

ACKNOWLEDGEMENT

The authors are highly thankful to the Head of the Microbiology department and the Bacteriology section in charge of Topiwala National Medical College and B. Y. L. Nair Charitable Hospital, Mumbai.

CONFLICT OF INTERESTS

Declared None

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