Evaluation of the Synergetic Effect of Water Soluble Extracts of Green Tea (Camellia sinensis) on the Activity of Ciprofloxacin in Urinary Isolated E. coli

MATERIALS AND METHODS

Bacterial isolates and culture media: A total of 18 isolates were collected from urine specimens submitted to a clinical diagnostic laboratory in Urmia, Iran during a two months period throughout October and November 2006. The isolates were further processed by the standard methods to identify as the Escherichia coli isolates. Isolated bacteria were maintained for long storage on skimmed milk medium (BBL) by adding 10% glycerol in-60°C, cultures were maintained for daily use on Nutrient agar slants on 4°C.

Preparation of tea leaves and ciprofloxacin powder: Commercial green tea was used for doing experiments. Tea samples were stored in plastic bags at 4°C. Crude green tea extracts were prepared by the method described by Tiwari et al. (2005) with some modifications. Ciprofloxacin powder were kindly provided by Exir Pharmaceutical Company, Tehran, Iran.

Determination of antimicrobial activity of green tea extracts: Antibacterial activity of water soluble green tea extracts and ciprofloxacin were measured by determining MIC and MBC for each bacterial isolate described previously else where (Jazani et al., 2007; Tiwari et al., 2005; Sahm and Weissfeld, 2002).

Determination of synergetic activity of green tea water extract with ciprofloxacin: Nutrient broth medium with green tea extract (initial concentration equivalent to MIC of each test isolate) prepared by two-fold serial dilution, ciprofloxacin were added to all tubes with sub-MIC concentrations. Each dilution was inoculated with 3x10⁶ of test isolate and incubated at 37°C. After overnight incubation, MIC and MBC of green tea in the presence of sub-MIC concentrations of ciprofloxacin were determined. (Tiwari et al., 2005). Synergy was present when there was a reduction in MIC of green tea extract in the presence of sub-MIC doses of ciprofloxacin for each strain (Gombert and Aulicino, 1983).

RESULTS

A total of 18 E. coli isolates were collected from urine samples submitted to the clinical microbiology laboratories of selected hospitals in Urmia, Iran.

Sensitivity of bacterial isolates to green tea water soluble extracts: The average MICs and MBCs of the water soluble green tea extract against all isolates of E. coli were 122.9±40.3 mg mL^-1. Also the MIC and MBC amounts were showed for each tested isolate (Table 1).

Table 1:	The MIC and MBC of ciprofloxacin, green tea water soluble extract and different concentrations of green tea extract in the presence of sub-MIC doses of ciprofloxacin for each E. coli isolate
*: MIC amount for each strain was equivalent to MBC, R = Resistant, S = Sensitive, NG = No Growth, NT = No Tested

Sensitivity of bacterial isolates to ciprofloxacin: As shown 4 isolates were resistant even to very high concentrations of ciprofloxacin investigated in this study, also there was 2 isolates that were sensitive to even very low concentrations (Table 1).

Synergism between water soluble green tea extract and ciprofloxacin: 43.7% of isolates showed decrease in MIC of green tea in the presence of sub-MIC concentrations of ciprofloxacin and 50% of E. coli isolates did not show growth in the presence of the sub-MIC concentrations of ciprofloxacin plus different concentrations of green tea extract under the MIC and for one of the isolates there is no difference in MIC of green tea extract in the presence or absence of sub-MIC concentration of ciprofloxacin, however 2 isolates did not tested in synergy experiments, because of their sensitivity to even very low concentrations of ciprofloxacin, there is synergistic activity between water soluble green tea extracts and ciprofloxacin between 93.7% of urinary tract E. coli isolates.

DISCUSSION

Escherichia coli is the most common cause of uncomplicated urinary tract infection and accounts for approximately 75 to 95% of all infections. Although the incidence of urinary tract infection has not changed substantially over the last 10 years, the diagnostic criteria, bacterial resistance patterns and recommended treatment have changed. Trimethoprim-sulfamethoxazole has been the standard therapy for urinary tract infection; however, E. coli is becoming increasingly resistant to medications. Many experts support using ciprofloxacin as an alternative and, in some cases, as the preferred first-line agent. However, others caution that widespread use of ciprofloxacin will promote increased resistance (Mehnert-Kay, 2005). Because of these reasons, we need new treatment modality that could replace traditional antibiotic therapy. Green tea components show antiviral and antifungal activities in addition to their inhibitory effects on exotoxins (Taguri et al., 2004). Various studies have shown significant suppressive effects of green tea against many microorganisms, for example Salmonella typhimurium (Shetty et al., 1994), Salmonella typhi, Shigella dysenteriae, Yersinia enterocolitica, Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Campylobacter jejuni, Plesiomonas shigelloides, P. aeruginosa and many other species of bacteria (Toda et al., 1989; Taguri et al., 2004; Yam et al., 1997, 1998; Kim et al., 2004; Stapleton et al., 2004; Lee et al., 2003). There is several evidence that high levels of green tea polyphenols were found in urine after drinking tea in human or experimental animal models (Kim et al., 2000; Yang et al., 1998). So using of green tea seems reasonable for treatment of urinary tract infections.

It has been shown that green tea has synergistic activity with some of antibiotics on different bacterial species especially E. coli and Staphylococcus aureus. Green tea catechins acting in synergy with beta-lactams, tetracycline and fluoroquinolones (Roccaro et al., 2004; Isogai et al., 2001).

In this study urinary tract isolates of E. coli were sensitive to green tea water soluble extracts, present results showed that green tea extracts had bacteriostatic as well as bacterioside effects on urinary tract isolates of E. coli (Table 1).

It has been shown that catechin (an extract of green tea) is an effective material in chronic bacterial prostatitis treatment in rat model. Synergistic effects between catechin and ciprofloxacin on chronic bacterial prostatitis rat model formerly has been proven by Lee et al. (2005). They create an experimental chronic bacterial prostatitis model in 70 male rats by instillation of 0.2 mL bacterial suspension of only one strain of E. coli (Z17, O2:K1:H). However in the present study we used different urinary tract isolates of E. coli obtained from patients. In this study in vitro synergistic effect of a combination of water soluble green tea extract and ciprofloxacin has been showed (Table 1), so present results confirms the data obtain from the study has been done by Lee et al. (2005) about Synergistic effects between green tea and ciprofloxacin on different urinary isolates of E. coli in vitro. Therefore, we suggest that the combination of green tea and ciprofloxacin may be effective in treating of urinary tract infections, However more studies on numerous strains of urinary tract isolates of E. coli as well as in vivo studies not only in experimental animals but also in humans should be done for confirmation of the synergistic antibacterial effects of water soluble green tea extracts in combination with ciprofloxacin for treatment of urinary tract infections caused by E. coli.

ACKNOWLEDGMENT

This study has been supported by a research grant from Student Research Committee of Urmia Medical Sciences University.