|Year : 2019 | Volume
| Issue : 1 | Page : 33-38
Evaluation of the antimicrobial property of green tea extract and its synergistic effect on antimicrobials showing resistance in clinical isolates of a tertiary care hospital
Vidushi Chaturvedi1, Shilpa Navinchandra Kaore1, Navinchandra Motiram Kaore2, Surender Kaur2, Shantaram K Gautam2
1 Department of Pharmacology, Raipur Institute of Medical Sciences, Raipur, Chhattisgarh, India
2 Department of Microbiology, Raipur Institute of Medical Sciences, Raipur, Chhattisgarh, India
|Date of Web Publication||14-Mar-2019|
Dr. Shilpa Navinchandra Kaore
Department of Pharmacology, Raipur Institute of Medical Sciences, Off NH-6, Bhansoj Road, Gam Godhi, Raipur - 492 101, Chhattisgarh
Source of Support: None, Conflict of Interest: None
Background: Antimicrobial resistance is a global health challenge with the increasing drug-resistant organisms even in community. Hence, there is a need for search of other alternatives to the antimicrobials which can replace or boost the activity of existing antimicrobials. Camellia sinensis (C. sinensis) is one of the most popular beverages worldwide and has been reported to demonstrate the antimicrobial activity against various pathogenic bacteria. Aim: The aim is to evaluate the antimicrobial property of green tea extract and its synergistic effect on antimicrobials showing resistance in clinical isolates. Materials and Methods: This cross-sectional, prospective, analytical study was conducted after due approval from the Institutional Ethics Committee. Green tea was procured and shade dried, and extraction was done by percolation and infusion method. The antimicrobial activities of these extracts were studied against standard strains and then on nonrepetitive clinical isolates from bacteriology laboratory showing resistance to the primary line of antimicrobials. Results: Our study showed antimicrobial action of green tea extract by percolation method against standard strain of Pseudomonas aeruginosa but not against Escherichia coli or Staphylococcus aureus. Minimum inhibitory concentration and minimum bactericidal concentration of green tea were found to be 12.5 μg/ml against standard strain of P. aeruginosa. The green tea extract exhibited synergistic activity against 21/30 clinical strains of nonfermenters tested from various clinical samples. No antibacterial action was found for extract prepared by infusion method against the standard American Type Culture Collection strains. Conclusion: C. sinensis could be explored as one of the alternatives or for synergistic action in combination with other antimicrobials for nonfermenters. Further investigation for potential toxicity and pharmacodynamic profile of the polyphenols is required.
Keywords: Aqueous extract, nonfermenters, phytophenols, Pseudomonas aeruginosa
|How to cite this article:|
Chaturvedi V, Kaore SN, Kaore NM, Kaur S, Gautam SK. Evaluation of the antimicrobial property of green tea extract and its synergistic effect on antimicrobials showing resistance in clinical isolates of a tertiary care hospital. J Mahatma Gandhi Inst Med Sci 2019;24:33-8
|How to cite this URL:|
Chaturvedi V, Kaore SN, Kaore NM, Kaur S, Gautam SK. Evaluation of the antimicrobial property of green tea extract and its synergistic effect on antimicrobials showing resistance in clinical isolates of a tertiary care hospital. J Mahatma Gandhi Inst Med Sci [serial online] 2019 [cited 2019 May 25];24:33-8. Available from: http://www.jmgims.co.in/text.asp?2019/24/1/33/254134
| Introduction|| |
Antimicrobial resistance is a global health challenge with the increasing drug-resistant organisms even in community. The overall cost burden on the health system with increased stay in the hospitals warrants urgent attention to the problem. With no new drugs in the pipeline, it is high time to search for some alternatives to the antimicrobials which can replace or boost the activity of existing antimicrobials.
Camellia sinensis (C. sinensis) is one of the most popular beverages worldwide and has been reported to demonstrate the antimicrobial activity against various pathogenic bacteria including resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa.,,,
The components in green tea that are responsible for these various pharmacological effects are polyphenols or also known as catechins. The main catechins in green tea are epicatechin (EC), EC-3-gallate (ECG), epigallocatechin (EGC), and EGC-3-gallate (EGCG), which constitute approximately 30%–40% of the water-soluble solids in brewed green tea.
Three of these catechins, ECG, EGC, and EGCG have been shown to have antimicrobial effects against a wide variety of microorganisms., The two polyphenols found in the highest amounts in green tea are EGC and EGCG. Both of these are excreted in bile, but EGC is also excreted in the urine, suggesting the possibility for green tea having antimicrobial activity against uropathogens.,
Being popular beverage routinely consumed by general population now-a-days, this study was aimed to establish the antimicrobial activity of the green tea extract against the standard strains and testing the optimal concentration of the same for synergistic activity in various clinical isolates showing multidrug resistance toward many of the primary antimicrobials used.
| Materials and Methods|| |
This cross-sectional, prospective, analytical study was carried out in the department of pharmacology and microbiology of a teaching institute attached to tertiary care hospital in central India over a period of 2 months from July 1st to August 31st 2018 after due approval from the Institutional Ethics Committee. The aqueous extract of green tea was tested against the standard strains of bacteria and later for synergistic activity on nonrepetitive clinical strains from patients of all ages and genders showing resistance to the primary line of antimicrobials used on routine antimicrobial testing. All repetitive samples as well as fungal and mycobacterial isolate were excluded from the study.
Preparation of green tea extract by standard percolation method
Green tea leaves identified by a botanist were shade dried and powdered. Two hundred milliliters of the boiling water was poured over 50 g of the powdered leaves and soaked overnight. The solution was percolated with addition of sterile distilled water till total extraction indicated by colorless extract getting percolated. Extract was then further concentrated using the water bath to achieve a concentration of 200 μg/ml.
Preparation of green tea extract by infusion method
To look for the activity of green tea extract the way we regularly drink it, we have prepared and decided to test the activity of extract prepared by simple infusion with a hypothesis that taking oral medication with green tea infusion extract may help to potentiate the activity of the antimicrobial taken.
To prepare infusion extract, 5 g of the green tea which was shade dried and powdered was taken in a sterile glass funnel over filter paper no. 1. Fifty ml of the boiling was poured over the powder. The infusion was collected in a sterile calibrated conical flask which was 19 ml with concentration of 270 mg/ml.
Antimicrobial activity on standard strains
The prepared extract was tested on the standard American Type Culture Collection (ATCC) strains of the E. coli 25922, S. aureus 25923, and P. aeruginosa 27853 a following the Clinical and Laboratory Standards Institute (CLSI) guidelines 2017 by disk diffusion method. Stains were tested against doubling dilution of the extract prepared by percolation, i.e., 200 μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, and 6.25 μg/ml along with the positive and negative controls. Similar double dilutions were also prepared of the infusion extract.,
The Mueller-Hinton agar (MHA) plates were lawn cultured with the standard strains after matching the broth prepared with the 0.5-McFarland (MF) turbidity standard. The disk of primary drug was placed as positive control (PC). A total of 6 wells were cut of 2 mm and filled with the 20 μl of serially double-diluted green leaf extract prepared in deionized water. A well with a sterile deionized water served as negative control (NC). The plates were incubated at 37°C for 18–24 h and were looked for the zone of inhibition (ZoI) showing similarity to standard drug used.,
Minimum inhibitory concentration
For determining the minimum inhibitory concentration (MIC), the green tea extract was double diluted in sterile peptone water as a noninhibitory media to prepare concentrations ranging from 200 μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, to 3.12 μg/ml in sterile test tubes. Uninoculated peptone water was used as NC, whereas inoculated peptone water was used as PC. The standard strain against which the MIC needs to be evaluated was inoculated in peptone water and incubated for 3–4 h to be in log phase. The inoculum was adjusted to 0.5-MF turbidity standard. Fifty μl of the inoculum was put in all the green tea extract dilution ranging from 200 to 3.12 μg/ml including PC. NC was left uninoculated. All the test tubes were incubated at 37°C for 18–24 h in a BOD incubator and were observed for the highest dilution which is not showing any growth in the form of turbidity as in PC. The highest dilution (lowest concentration of green tea extract) which shows the inhibition of the growth was considered the MIC of that strain for green tea extract.,
Minimum bactericidal concentration
After doing the MIC as above, the concentration of green tea extract showing the MIC (no turbidity) and a subsequent two double dilutions of it (showing the growth as turbidity) were subcultured on noninhibitory media of brainheart infusion agar for any visible growth on the plating media. The highest dilution (lowest concentration of green tea extract) which shows no growth on the noninhibitory plating media was considered as the minimum bactericidal concentration which kills the test strain organisms inoculated during MIC.
Testing on the clinical isolates
Any of the clinical isolates showing resistance to any of the primary drugs tested in routine antibiotic sensitivity test according to the CLSI 2017 was tested further. The strains were lawn cultured on MHA in duplicate after matching with the 0.5MF standard turbidity. The primary drug line showing resistance was again tested on one plate to rule out any ambiguity. The second plate was with the antimicrobials showing resistance with addition of 20 μl of the green tea extract in the highest concentration. The plates were incubated at 37°C for 18–24 h and were looked for the increase in size of the ZoI, indicating synergism. The comparative evaluation of the zone size with the standard zone size as per the CLSI guideline 2017 was done to assess the effectiveness in vitro.,
All data were maintained in Microsoft Office Excel, and statistical analyses were carried out using tests of proportion.
| Observations and Results|| |
The aqueous extract of the green tea was evaluated against the standard strains of E. coli 25922, S. aureus 25923, and P. aeruginosa 27853 as a representative strain for various in vitro antimicrobial activity parameters, as shown in [Table 1].
|Table 1: In vitro activity of green tea extract by percolation method and infusion method|
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As shown in [Table 1], there was no activity against any concentration for E. coli 25922 and S. aureus 25923 with no ZoI on MHA, whereas P. aeruginosa 27853 showed good activity with ZoI ranging from 16 to 9 mm for 200–12.5 μg/ml, whereas no activity was seen for 6.25 μg/ml [Figure 1].
|Figure 1: Well marked Zone of Inhibition for P. aeruginosa for various concentrations used from 200 to 12.5 μg/ml|
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The infusion extract did not show any activity against any of the standard strain tested, so no further MIC and minimum bactericidal concentration (MBC) evaluation was done for the same.
As the activity was only seen with percolated extract in P. aeruginosa 27853 (PA), only this strain was tested further for MIC and MBC. As seen clearly, the standard strain PA showed MIC of 12.5 μg/ml with no growth with visible turbidity in higher concentrations [Figure 2].
|Figure 2: MIC of Green Tea Extract against P. aeruginosa showing MIC value of 12.5 μg/ml which shows no turbidity whereas 6.25 μg/ml showing the turbidity with Positive control at left end and Negative (un-inoculated) control at right end|
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The concentration range from 12.5 to 3.12 μg/ml was then subcultured on brain–heart infusion agar, and no growth was observed in 12.5 μg/ml, whereas growth of PA was seen when subcultured from 6.25 to 3.12 μg/ml.
The comparison of ZoI against the concentration of the green tea extract prepared by the percolation method is shown in [Graph 1].
Being effective against the ATCC strain of P. aeruginosa 27853 (PA), the percolated green tea extract was further tested against the clinical isolates of nonfermenters isolated in bacteriology laboratory attached to a tertiary care hospital. All the nonfermenters isolated and found resistant to any of the standard primary drugs as advocated by CLSI were further tested for enhancement or accentuation of activity indicated by the increase in ZoI by addition of the extract.
A total of 30 nonfermenting clinical isolates received in bacteriology laboratory were tested. Out of 30, 21 (70%) has shown enhancement of the ZoI suggesting a synergistic activity with the green tea extract. The average increase in the zone size with the 21 isolates showing enhancement was 4.80 mm [Graph 2].
| Discussion|| |
Tea is the most widely and popularly used daily beverage and crude medicine in China for thousands of years. It is a safe and cheap beverage reported to have antimicrobial activity against wide variety of organisms. The polyphenols are the most abundant group of compounds in fresh tea leaves found in green and black tea beverages. Pharmacological studies have shown tea to possess antimicrobial, antipyretic, diuretic actions; antioxidative efficacy; antihypercholesterolemic activity; and antimutagenic, anticancer, and antitumorigenic effects.
Resistance mechanisms acquired and expressed by the pathogenic organisms against variety of the antimicrobials in use pose a difficulty in treatment of the infections they cause. If it is exhibited to a large number of antibiotics, treatment becomes progressively very difficult. Therefore, it seems reasonable to explore new sources of natural compounds with antibacterial activity against them. C. sinensis has been proved to possess medicinal and health promotion properties, including the ability to inhibit the growth of some types of pathogenic bacteria. The observed effects of combinations of flavonoids and medicinal antibiotics are beneficial. However, its utility in the prevention and therapy of human infections awaits approval. If they are indeed effective, patients would be exposed to lower levels of antibiotics, thus minimizing any side effects and arresting or delaying development of antibiotic resistance.
Gram-positive organisms exhibit variety of resistance mechanisms such as beta-lactamases, methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus, vancomycin-resistant enterococci, and inducible clindamycin resistance, whereas Gram-negative organisms possess beta-lactamases, extended-spectrum beta-lactamases, AmpC beta-lactamases, metallo-beta-lactamases, and latest carbapenemase-producing Enterobacteriaceae. These varied spectrums of drug resistance mechanisms are exhibited by variety of Gram-positive and Gram-negative organisms, which cause infection in hospitalized patients, causing a number of nosocomial infections such as bacteremia, urinary tract infections, and nosocomial pneumonia. Nowadays, the resistant strains are also encountered in the infections from community (community acquired).
In our study, we explored antimicrobial activity of green tea extract on standard E. coli 25922, S. aureus 25923, and P. aeruginosa 27853 following the CLSI guidelines 2017 by disk diffusion method, since these were taken as representative of Gram-negative, Gram-positive, and nonfermenters, respectively. The green tea extract concentrations used were 200 μg/ml and progressively double diluted six times, i.e., 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, and 6.25 μg/ml. There was no activity against any concentration for E. coli 25922 and S. aureus 25923 with no ZoI on MHA, whereas P. aeruginosa 27853 showed good activity with ZoI ranging from 16 to 9 mm for 200 μg/ml–12.5 μg/ml, whereas no activity was seen for 6.25 μg/ml. The average increase in the zone size with the 21 isolates showing enhancement was 4.80 mm from a total of 30 isolates. The infusion extract has not shown any activity against any of the standard strain tested, so no further MIC and MBC evaluation was done for the same.
The result of our study is in accordance with other studies that have previously reported the antibacterial activity of tea against resistant strains of P. aeruginosa. In a study conducted by Maksum Radji et al., in 2013, ATCC strains of S. aureus and P. aeruginosa were tested for MIC which came out to be 400 and 800 μg/ml. The zone size inhibition with green tea extract was found to be 18.970 ± 0.287 and 19.130 ± 0.250 for ATCC and MRSA strains of S. aureus, respectively, whereas for ATCC strain of P. aeruginosa and multidrug-resistant (MDR) P. aeruginosa, it was found to be 17.550 ± 0.393 and 17.670 ± 0.398, respectively.
According to the study by Jazani et al., the average of MICs and MBCs of the water-soluble green tea extract against all strains of P. aeruginosa (of which 55.8% were MDR strains) was 2.06 ± 1.76 and 2.54 ± 2.22 mg/mL; while in our study MIC and MBC was found to be 12.5 μg/ml. Several previous studies have shown that green tea extract showed activity against MDR P. aeruginosa, as demonstrated in our study, while the activity was also demonstrated against MRSA and methicillin-sensitive S. aureus, in contrast to our study. In a study done by Yi et al., ultrastructural analysis provided evidence that tea polyphenols (TP) caused alteration in the integrity of the membranes and disruption of cell walls causing permeability and leakage of intracellular components.
In study by Chan et al., various green, black, and herbal tea extracts were evaluated against three Gram-positive organisms, namely Micrococcus luteus, S. aureus, and Bacillus cereus, where green teas inhibited all the three Gram-positive bacteria, including S. aureus, which is in contrast to our study. They also evaluated antimicrobial activity for three Gram-negative bacteria such as E. coli, Salmonella More Details typhi, and P. aeruginosa and found that none of the tea extracts showed any activity against these. In addition, they found that black and herbal teas inhibited the growth of M. luteus and B. cereus but not S. aureus.
Another study using aqueous, ethanolic and methanolic extracts of tea have concluded that aqueous extracts show minimum antibacterial activity, whereas maximum activity is exhibited by methanolic extracts and exhibited significant activity against E. coli and S. aureus. This is in contrast to our study for aqueous extract, and we have not evaluated the ethanolic or methanolic extract. Their study reported least activity against Pseudomonas and Proteus, whereas in our study, we have found good antimicrobial activity of aqueous green tea extract against P. aeruginosa.
Studies show the highest antimicrobial activity corresponds to the highest antioxidant activity shown by Trolox equivalent antioxidant capacity (TEAC) values and less to total polyphenol content. According to the international journal of food properties, a study was conducted through high-performance liquid chromatography to determine qualitative and quantitative green tea catechins which gave the highest recovery of EGC, EGCG, ECG, and EC with extraction time of 40 min. Results also confirmed that among the individual catechins, EGCG was present in the highest quantity and EC was in the lowest values (EGCG > EGC > ECG > EC).
The infusion extract has not shown any activity against any of the standard strains tested in the present study. Daljit et al. (2009) reported that the 5% aqueous green tea extract can also inhibit the growth of P. aeruginosa because the total phenolic contents, especially EGCG, exert antimicrobial activity on P. aeruginosa., The variation of the response of antimicrobial action with different studies might be presumably due to different methods of processing of tea, variation in species of tea procured, their seasonal variation and collection process, and last but not the least, the mode of drying.
In contrast to our studies, wide range of nonresistant pathogenic organisms by aqueous extracts of green tea has been reported to have shown synergistic activity with aqueous extract.,, Theasinensin A is a decomposition product of EGCG that has been reported to prevent antibiotic resistance of MRSA. EGCG at MIC values of < 100 μg/ml has also been reported to reverse resistance of Staphylococci to tetracycline. Combinations of various teas with gentamicin, methicillin, and nalidixic acid revealed synergistic action against Shigella dysenteriae. Green tea extract has also been reported to show synergistic activity with antibiotic such as chloramphenicol, amoxicillin, sulfamethoxazole, azithromycin, levofloxacin, methicillin, nalidixic acid, gentamicin, and ciprofloxacin against E. coli.
Although EGCG reaches detectable levels in serum following oral administration of green tea powder, concern has been raised about the potential for toxicity when administered at high dose.
It can be thus suggested that C. sinensis leaf extract can be used as complementary medicine in treating diseases caused by MDR strains of P. aeruginosa. However, further investigation is needed to determine the bioavailability of the active compounds and to determine the dose and toxicity before it can be used as therapeutic agents.
| Conclusion|| |
The antibacterial activity of C. sinensis leaves was explored in this study against standard ATCC strains and pronounced antibacterial action was found against P. aeruginosa exerted through its polyphenolic constituents. The synergistic activity against clinical strains of nonfermenters has been demonstrated. Further investigation for potential toxicity and pharmacodynamic profile of the polyphenols would explore more potential for its use in general.
We would like to acknowledge the ICMR for choosing the important topic under short-term studentship program. We would like to extend our thanks to Mr. Khomlal Sahu, Technician Microbiology Department, and Mr. Jitendra, Clerk in Pharmacology, for all the technical and official helps extended in carrying out the study.
Financial support and sponsorship
This study was self funded with grant from STS-ICMR and supported by the Department of Microbiology and Pharmacology, Raipur Institute of Medical Sciences (RIMS), Off NH-6, Bhansoj Road, Gam Godhi, Raipur - 492 101, Chhattisgarh, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]