MATERIALS AND METHODS

The samples were collected from CSI, Mission General Hospital, Woraiyur, Tiruchirappalli, Tamilnadu, India, during December-2004 to February-2005. Midstream urine samples were collected from 85 female patients and 42 male patient in aseptic condition.

Preparation of plant extracts: Leaves of both the species were collected from mature plants and identified by comparing with herbarium specimens. The leaves were air- dried and powdered. The dry leaf powder was extracted by reflexed in 100 mL methanol for 24 h, using a Soxhlet apparatus (Khan et al., 1988). The extract was filtered using Whatman filter paper, No. 1. The filtrate was then evaporated using rotatory evaporator and dried at 55°C. Dried extract was stored at 20°C in labeled, sterile screw-capped bottles.

Microorganisms: The bacterial colonies were isolated from hospital samples, their pure cultures were maintained in nutrient agar and stored at 4°C. Ten bacterial species were isolated, namely E. coli. Klebsiella pneumonia, Salmonella typhi, Salmonella paratyphi A, Staphylococcus aureus, Methicilin Resistant Staphylococcus aureus (MRSA), Edwardsiela tarda, Pseudomonas aeruginosa, Citrobacter diverses, Serratia liquefaciens and the fungus Candida albicans.

Antimicrobial assay: Sensitivity tests were performed by disc diffusion with standard antibiotics, following Kirby-Bauer method (Bauer et al., 1966). The air-dried extracts at different concentrations such as 10, 20, 30, 40 μL were dissolved in DMSO and loaded in empty sterile discs. The assessment of antibacterial activity was based on measurements of the diameter of inhibition zones (NCCLS, 1998).

RESULTS

Antimicrobial activity of the methanolic extracts of Clitoria ternatea was tested against eleven urinary tract pathogens. Table 1 shows the inhibitory effect on seven urinary pathogens: Klebsiella pneumonia, Salmonella typhi, Salmonella paratyphi, Staphylococcus aureus, Methicilin Resistant Staphylococcus aureus (MRSA), Edwardsiela tarda and a fungus Candida albicans. The extracts had no inhibitory effect against E. coli, Pseudomonas aeruginosa, Citrobacter diverses and Serratia liquifaciens. Among the different concentrations used, 40 μg concentration showed maximum activity against the susceptible pathogens. Maximum inhibitory zone was formed against Edwardsiella tarda and Candida albicans. The two microbes were highly susceptible and showed 14 mm zone of inhibition. A zone of inhibition of 12 mm was observed for Klebsiella pneumoniae and MRSA.

Methanolic extract of Achyranthes aspera showed effective antimicrobial activity against four urinary pathogens: Klebsiella pneumonia, staphylococcus aureus, Salmonella paratyphi and Methicilin resistant Staphylococcus aureus.

Among the ten microbes tested, two gram positive, five gram negative bacteria and a fungus were susceptible to the methanolic extract of Clitoria ternatea. Methanolic extract of Achyranthes aspera also showed similar antimicrobial effect, however to a lesser extent against the UTI pathogens. It showed antimicrobial activity against four pathogens, namely Klebsiella pneumonia, Salmonella paratyphi-A, Statphylococcus aureus and MRSA. Antimicrobial activity of Clitoria ternatea and Achyranthes aspera were comparable to those by standard antibiotics, although at a lesser degree in overall inhibitory activity. The UTI bacterial pathogens were resistant against nystatin, only Candida albicans showed zone of inhibition. Amikacin showed maximum inhibition against all pathogen near 20 mm, whereas vancomycin and penicilin recorded maximum resistance from all pathogen and recorded lowest zone of inhibiton of 8 mm. Even methanolic extracts of the two plants exhibited more zone of inhibition against MRSA, Salmonella typhimurium, Salmonella paratyphi-A when compared to standard discs of Amikacin, Penicilin and Vancomycin (Table 2).

Table 1:	Survey of antimicrobial activity of Selected plants
1: E.coli, 2: Klebsiella pneumoniae, 3: Salmonella typhimurium, 4: Salmonella paratyphi- A, 5: Staphylococcus aureus, 6:Methicilin Resistant Staphylococcus aureus, 7: Edwardsiella tarda, 8: Pseudomonas aeruginosa, 9: Citrobacter diverses, 10: Serratia liquifaciens, 11: Candida albicans

Table 2:	Antimicrobial activities of Standard antibiotics (Inhibition zones in mm)
A: Ampicillin, Ctx: Cefataxime, Va: Vancomycin, Ofx: Ofloxacin, P: Penicillin, Te:Teicoplanin, Ak-Amikacin, NY: Nystatin

DISCUSSION

Medicinal plants are used by large proportion of Indian population. The reasons for this include true improvement, absence of harmful side effects and the high cost of other forms of treatment. Urinary Tract Infections (UTIs) are a leading cause of morbidity and involve high health care expenditure in persons of all ages. Sexually active young women are disproportionately affected, but several other populations including elderly persons and those undergoing genito-urinary instrumentation or catheterization, are also at risk. An estimated 40 percent of women reports having had a UTI at some point in their lives. UTIs are the leading cause of gram-negative bacteremia. Several plants have been used in traditional medicines to combat UTI.

From the earlier studies it is obvious that most of the plants differ significantly in their antimicrobial property. These differences may be attributed to the differences in the cell wall constituents of bacteria which vary among the gram positive and gram negative ones (Yao and Moellering, 1995; Ozcelik, 1998). Inhibitory effect of medicinal plant extract exhibited against UTI pathogens in this study is similar to the results obtained in the following studies. In general, alcoholic extracts exhibit highest degree of antimicrobial activity as compared to aqueous and hexane extract fractions. The inhibitory activity was found to be maximum in the methanolic extracts of Euphorbiaceae plants against urinary tract pathogens such as E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (Ramesh et al., 2001). Antimicrobial activity against Klebsiella was observed in oils extracted from Ocimum and Cymbopogom (Pereira et al., 2004). Antimicrobial activity was also documented against Klebsiella pneumoniae and Staphylococcus aureus in the root extract of Pholidata articulata (Mehmood et al., 1999). Antibacterial activity against E.coli, Staphylococcus aureus and Pseudomonas aeruginosa was exhibited by Chlorophytum borivilianum (Sundaram et al., 2011). Similar zones of inhibition could be obtained in this study with the methanolic extracts Clitoria ternatea and Achyranthes aspera. Antimicrobial activity of these plants was thus evident.

Earlier studies have established that traditional medicinal plant extracts also produce effective inhibitory zones against Salmonella typhi and Salmonella paratyphi (Ahmad and Beg, 2001; Rani and Khullar, 2004). Similar results could also be obtained in the present experiment against those urinary pathogens by the extract of Clitoria ternatea. Antifungal activity exhibited was also compared to earlier reports with other plant extracts (Ahmad and Beg, 2001). Methanolic extracts of African medicinal plants also show similar results against all common pathogens similar to that observed in this study (Mariita et al., 2011).

Isolation of microbial agents less susceptible to regular antibiotics and recovery of increasingly resistant isolates during antibacterial therapy are rising throughout the world (Obrien et al., 1999; Cookson, 2000; Archibald et al., 1997). To combat multidrug resistant strains like MRSA, the development of new antibacterial compounds for substituting the ineffective ones is the immediate task. As medicinal plants as well as microorganisms are the appropriate candidates, they should receive continuous research attention. Further development of antibiotic resistant strains and the side effects caused by the overuse of antibiotics is a serious concern. Hence much attention is being paid recently towards extracts and biologically active compounds isolated from plants used in herbal medicine (Essawi and Srour, 2000; Cos et al., 2002; Shahidi et al., 2002). At this juncture it is interesting to note that both the plant extracts used, showed effective inhibitory zones against Methicillin Resistant Staphylococcus aureus (MRSA). Similar results was observed in the earlier study of Anam et al. (2010) using leaf extract of Terminalia muelleri, which showed antibacterial activity against MRSA closer to range of standard antibiotics discs of Vancomycin and Penicilin.

From the present study it is evident that the crude methanolic extracts of Clitoria ternatea and Achyranthes aspera possess antibacterial and antifungal property. Further identification and isolation of specific antimicrobial compounds and their purification can pave better ways to control these kinds of urinary tract infections, and also to find novel approaches to tackle the emergence of drug resistant strains of pathogens.