Hana Mohammed Ahmed*, Babiker Saad Almugadam, Shima Mubarak Mohammed Baher and Khansa Elneel Suliman Elbasher
Department of Microbiology, Faculty of Medical Laboratory Sciences, University of El Imam El Mahdi, Kosti city, Sudan
Received date: February 03, 2018; Accepted date: March 03, 2018; Published date: March 07, 2018
Citation: Ahmed HM, Almugadam BS, Baher SMM, Elbasher KES (2018). Antimicrobial Effect of Green Tea Extract on Uropathogenic Escherichia coli Isolates. J Pharm Microbiol. Vol.4 No.1:1.
Background: The development of antibiotic resistance in bacteria is a growing problem worldwide especially in uropathogenic Escherichia coli (E.coli) isolates.
Objective: This work was designed to assist the in vitro antimicrobial effect of green tea extract against uropathogenic E.coli isolates and to determine the MIC and MBC of green tea extract.
Materials and Methods: This is experimental laboratory based study. A total of 10 uropathogenic E.coli isolates were collected from urinary tract infected patients attended Kosti Teaching Hospital, Sudan during period from September to October 2017. All isolates were identified base on colonial morphology, Gram strain, and standard biochemical tests. Each isolate was subjected to evaluation of their susceptibility to green tea extract using well diffusion and broth dilution method. Data was analyzed using statistical package for social sciences (SPSS) software version 17.
Results: The means of inhibition zone of bacteria to 25%, 50%, and 100% concentrations of green tea extract were 10 mm, 18 mm, and 30 mm respectively. The MIC and MBC of green tea extract for most isolates were 200mg/ml and 400mg/ml respectively.
Conclusions: Green tea extract has in vitro antimicrobial effect on uropathogenic E.coli. Implementation of in vitro and in vivo studies to evaluate the antimicrobial effect of green tea and the efficacy of its catechins in the treatment of UTIs can lead to discover of a cheapest therapy.
Green tea extract, MBC, MIC, Uropathogenic E.coli, UTI
Urinary tract infections (UTIs) are the most common type of infection worldwide, and have resulted in billions of dollars in medical care costs [1,2]. E.coli is the most important cause of 80- 90% of all UTIs . Uropathogenic E. coli infects the urinary tract by producing special surface proteins (adhesions), which give them the ability to attack the epithelial cells that line the urinary bladder [4,5]. If pathogenic E. coli is in the bladder, and is not eliminated, it may travel up the ureters to the kidneys and cause pyelonephritis [3,6]. The development of antibiotic resistance in bacteria is a growing problem worldwide. A numbers of E.coli isolates have been collected from urine specimens of patients with UTI that are resistant to antimicrobial agents commonly used to treat UTIs [7,8]. Therefore, treatment options are replaced with a second or third choice of antibiotics, which are much more expensive . These challenges have been receiving growing interest to find alternative antimicrobial agents from plant extracts that need to be developed and use to control multidrug resistant bacteria [9,10]. Camellia sinensis (green tea) is one of the most popular beverages in the world and has been reported to have antimicrobial effects against various pathogenic bacteria . Tea can be cultivated in many regions from sea level to high mountains. It is generally safe nontoxic, cheap, and available. These properties make it a very good alternative antimicrobial agent. Many studies on the antibacterial activity have shown that green tea inhibits the growth of E. coli by it is polyphenolic components (also known as catechins). The bactericidal action of catechins is due to its hydrogen peroxide generation . Also Polyphenols are anti-inflammatory agents that inhibit clinical symptoms of UTIs . Catechins induce production of cytokines such as IL-12 and IL-10, blocking the connection of conjugated R plasmid in E. coli that have bactericidal and antitoxin effects, and decrease tumor necrosis factor alpha gene expression which is important in pathogenesis of E. coli infection . Catechin-copper II complexes damage the cytoplasmic membrane of E.coli . This work was designed to assist the in-vitro antimicrobial effect of green tea extract against uropathogenic E.coli isolates and to determine the minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of green tea extract against it.
This is experimental laboratory based study, carried out in the Department of Microbiology, Faculty of Medical Laboratory Sciences, University of El Imam El Mahdi in Kosti city, White Nile state. A total of 10 uropathogenic E.coli isolates were collected from urinary tract infected patients attended Kosti Teaching Hospital during period from September to October 2017. All isolates were identified based on colonial morphology, Gram stain, and standard biochemical test . Each isolate was subjected to evaluation of their susceptibility to green tea extract using well diffusion and broth dilution method. Data was analyzed using statistical package for social sciences (SPSS) software version 17.
Preparation of the plant extract
Obtained a green tea from the local markets in Kosti of dried leaves stored in the bags packed (this type of green tea is Chinese origin). Aqueous extract was prepared by mixing 50gm of Camellia sinensis non fermented leaves with 500ml distilled water in volumetric flask capacity of 1000 ml and left stuck with stirring in water bath for 24hours at a temperature of 40oC, later the extracts filtered through several layers of sterilized gauze first then through filter paper. Then the filtered extracts poured in clean and sterile petri dishes and left to dry in the oven at 50°C for 3 days. 25%, 50%, and 100% concentrations were prepared from the stuck of green tea extract using distal water .
Agar well diffusion method
It was done to evaluate the inhibitory effect of different concentrations of the green tea extract. Bacterial suspensions equivalent to 0.5 McFarland standards was prepared from each isolate. 0.1ml of the suspension was spread on MH agar using sterile cotton swab. Then pores with 5mm diameter were made by the piercing cork in the rate of 3 pores for each plate. 0.1ml of each concentration of the extracts laid down in pores. Each plate was incubated aerobically at 37°C for 24hours. The inhibition zones of each isolate to different concentrations of green tea extract were measured in mm [16,17].
Broth dilution method
It was used to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of green tea extract. The inoculums was prepared by making a direct broth suspension of isolated colonies using peptone water medium (from non-selective medium, incubation not more than 18-24 hour), then the suspension was adjusted to achieve turbidity equivalent to 0.5 McFarland turbidity standards. Different concentrations (25, 50, 100, 200, and 400mg/ml) of green extract were prepared by dissolve the powder of each concentration in 1ml of peptone water then 100μl of adjusted inoculum was added to each concentration, and to a positive control tube containing only broth. The procedure was done for each isolate. All tubes were mixed and incubated aerobically at 37oC for 24 hour. MIC and MBC were measured. MIC is a first concentration in the tubes showed non visible growth. MBC was detected by subculture of the tubes next to MIC tube and reported as first concentration has negative growth in subculture .
The means of inhibition zone of bacteria to different concentrations of green tea extract were displayed in Table 1. The MIC and MBC of green tea extract for most isolates were 200mg/ ml and 400mg/ml respectively as shown in Table 2.
|Diameter of zone (mm)||30||18||10|
Table 1 The means of inhibition zone diameter (mm) of bacteria for different concentrations of green tea extract.
|Isolate number||MIC (mg/ml)||MBC (mg/ml)|
Table 2 The minimum inhibitory concentration and minimum bactericidal concentration of green tea extract.
MIC and MBC of all isolates were 200mg/ml and 400mg/ ml respectively, except for isolate number 6 and 10. The MIC and MBC for number 6 and 10 was 100mg/ml and 200mg/ml respectively.
In the last several years, the frequent occurrence of infections with beta-lactamases producers has been observed globally. The advent of beta-lactamases producers has established a great hazard to the use of many classes of antibiotics particularly cephalosporin. The high costs of treatments make attention to importance of alternative cheaper therapy. In this regard, plant extracts are being comprehensively researched by scientists. Green tea is consumed worldwide now and its beneficial physiological and pharmacological effects are very well known. As we reported in this study the means of inhibition zone of bacteria to 25%, 50%, and 100% concentrations of green tea extract were 10 mm, 18 mm, and 30 mm respectively. And the MIC and MBC of green tea extract for most isolates were 200mg/ml and 400mg/ ml, respectively. Our data suggests that the green tea extract has highly in vitro antimicrobial activity against uropathogenic E.coli isolates that strongly consistent with the results of Passat et al. study that reported, green tea water extract has antimicrobial effect on E.coli strains with MIC 275 mg/ml, which is slightly low when compare with our study . Also our study agrees with Tiwari et al. study that reported green tea extract has antimicrobial effect against E.coli, while we disagree with Tiwari et al. study in the value of MIC as they reported it is 88.30mg /ml. The main cause of difference in the value of MIC between Tiwari et al. study and our study may be the use of green tea extract combined with ciprofloxacin in Tiwari et al. study .
Green tea extract has in vitro antimicrobial effect on uropathogenic E.coli. Implementation of in vitro and in vivo studies to evaluate the antimicrobial effect of green tea and the efficacy of its catechins in the treatment of UTIs in future can lead to discover of a new cheaper therapy region.
Authors would like to thanks participant patients and staffs of the Department of Microbiology, University of El Imam El Mahdi. The study was approved by Ethic Review Committee, University of El Imam El Mahdi.