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Short Communications Assessment Of Short Communications Pakistan J. Zool., vol. 45(2), pp. 555-558, 2013. diarrohoea, endocarditis, and bacterimia (Nannini et al., 2005). Enterococci are facultative anaerobic, Assessment of Antibacterial Activity Gram positive cocci that live as normal flora in the of Momordica charantia Extracts and gastrointestinal tract of humans and animals (Kiem et al., 2003). Enterococcus species are indicators of Antibiotics against Fecal animal and human fecal contamination in water and Contaminated Water Associated various food products (Moneoang and Enterococcus spp. Bezuidenhout, 2009; Valenzuela et al., 2008). More than twenty species of Enterococci Saiqa Andleeb1*, Tahseen Ghous2, Nazia Riaz1, have been classified. Enterococcus faecium and Nosheen Shahzad2, Summya Ghous1 and Uzma Enterococcus faecalis are the mostly indentified Azeem Awan1 species in humans, animals, food products and water 1Biotechnology laboratory, Department of Zoology, (Facklam, 2002). Fisher and Philips (2009) Azad Jammu and Kashmir University, demonstrated that these pathogens would cause Muzaffarabad 13100, Pakistan disease if the hosts immune system is suppressed. 2Biochemistry Laboratory, Department of Hydrogen peroxide derived from E. faecium was Chemistry, Azad Jammu and Kashmir University, shown to damage luminal cells in the colon of rats Muzaffarabad 13100, Pakistan (Huycke et al., 2002). Infectious pathogens have been reduced using various medicinal plants such as Abstract.- Antibacterial activity of Momordica charantia due to their potential extracts of Momordica charantia and several antidiabetic, antihelmintic, antmicrobial, anti- antibiotics were studied against Enterococcus faecalis and Enterococcus faecium isolated cancerigenos and antioxidant activities (Costa et al., from water receiving fertilizers of animal origin 2011). In the present study Enterococcus pathogens by filter disc diffusion method. E. faecalis and were isolated from animal fecal contaminated water, E. faecium were found sensitive to gentamycin, causing infections in human as well as in other ciprofloxacin and tetracycline, and resistant to mammals, to determine the pattern of antibacterial penicillin G, trimethoprim and sulfometho- xyzol. M. charantia showed significant zone of resistance. The aim of present work is to determine inhibition in the case of n-hexane, ethanol and the antibacterial activity of M. charantia and to methanol extracts of green parts against A and assess the efficacy of different antibiotics in use C strains of E. faecium, while ethyl acetate and against E. faecium and E. faecalis. methanol extracts of seed parts of M. charantia indicated considerable inhibitory effect against both B and A strains of E. faecium. This study Materials and methods showed that fruit part of M. charantia could be Sample collection a potential source of new antimicrobial agents. Water samples were collected from the different areas of contaminated river near Muzaffarabad city, Key words: Agar disc diffusion method, antibiotics, antibacterial activity, Enterococci, Pakistan. A sample of 200 µl was spread on Slantez Momordica charantia. and Bartely medium (SBM) plates (Slanetz and Bartley, 1957), and incubated at 37oC for two days. nterococci are considered part of the E Isolation and identification of Enterococcus normal flora of food products, water, bowel, Appearance of growth on SBM proved the mammals, birds and urethra of humans (Reeves and presence of the members of genus Enterococcus. Grant, 2004). Although, Enterococci are of Two types of Enterococcus viz., E. faecalis and E. relatively low virulence, opportunistic pathogens faecium were identified and confirmed by can cause serious humans diseases including conventional microbiology and biochemical ____________________________ procedures performed in the Microbiology lab of * Corresponding author: [email protected] 0030-9923/2013/0002-0555 $ 8.00/0 Combined Military Hospital, Muzaffarabad, Copyright 2013 Zoological Society of Pakistan Pakistan. Screening of pathogens were carried out 556 SHORT COMMUNICATIONS by using various growths promoting bacterial media filter paper disc (5 mm diameter) was impregnated i.e., MacConky agar, nutrient agar, nutrient broth, with crude plant extracts and placed on nutrient agar brain heart infusion agar, thiosulphate citrate-bile which was previously swabbed with pathogens. salts- sucrose agar, and eosin methylene blue agar Antibiotics were also used to check the purchased from Oxoid. The Gram staining was done susceptibility test. The methanol, ethanol, to characterize the isolates. Various biochemical chloroform, ethyl acetate, and n-Hexane were used tests such as oxidase, catalase, coagulase, and API as blind controls. Finally the inoculated plates were 10 were used for confirmation of pathogens (Clarke, incubated for 24 h at 37°C to allow the maximum 1953; Slanetz and Bartley, 1957). growth of the microorganisms and the zone of inhibition was observed and measured in Preparation of extracts of medicinal plants millimeters. Each assay in this experiment was M. charantia, purchased from the super repeated for three times. Growth inhibition (GI) was market of Muzafarrabad, was air dried at room recorded as very high (++++), high (+++), medium temperature for 10 days and then powdered with the (++) and low (+), which indicated zones of help of a blender. The extracts of M. charantia (both inhibition between 35-49, 21-34, 12-20, below 12 seeds and green parts) were prepared consecutively mm, and no activity indicated as zero mm. GI was with, n-hexane (N1, N6), chloroform (N2, N7), measured as “zone of inhibition= inhibited area-disc ethyl acetate (N3, N8), ethanol (N4, N9) and size”. methanol (N5, N10) using a Soxhlet extractor for 48 h. All the extracts were concentrated using rotary Results and discussion flash evaporator and preserved at 4°C in airtight Previous studies demonstrated that medicinal bottle until further use. All the extracts were tested herbs are a new potential source of antibacterial for antibacterial activity assay. The extracts of seeds agents even against some antibiotic-resistant strains indicated as N1, N2, N3, N4, N5 whereas green (Kone et al., 2004). Results of this study confirmed parts of M. charantia as N6, N7, N8, N9, and N10, the observation of earlier studies (Yuste and Fung, respectively. 2004). Enterococcus is Gram positive bacterium, Sensitivity test of standard antibiotics API 10, carbohydrate, and indole positive but Sensitivity of antibiotics against isolated negative for catalase test (Clarke, 1953; Slanetz and pathogens was determined by filter paper disc Bartley, 1957). Three different strains of E. faecium method (Prescott et al., 1990). Sensitivity was (A, B, C) and one strain of E. faecalis (D) were predictable with clear zone surrounding the disc. identified. The potency of antibiotics per disc (5 mm in Table I shows the effect of various antibiotics diameter) was as follows: amoxicillin (10 µg), on multiple drug resistant Enterococcus species streptomycin (10 µg), tobramycin (10 µg), (Courvalin, 2006). All the tested pathogens were gentamycin (10 µg), ciprofloxacin (5 µg), resistant to pencilline G, trimethoprim and sulfomethoxyzol (25 µg), tetracycline (10 µg), sulfomethoxyzol, and sensitive to tetracycline, penicillin G (10 µg), trimethoprim (5 µg), ampicillin ciprofloxacin, and gentamycin (Table I). It was (10 µg). observed that E. faecalis was sensitive to gentamycin, ciprofloxacin and tetracycline and Filter paper disc diffusion method resistant to remaining antibiotics (Table I). Our Filter paper disc method was used for testing results are consistent with Arias et al. (2010) and medicinal plant extracts against the selected Hooper and Wolfson (1991). Likewise, E. faecium pathogens (Alzoreky and Nakahara, 2003). The (B) was sensitive to amoxylin, ampicillin, Nutrient agar was prepared, allowed to cool up to 40 gentamycin, ciprofloxacin, and tetracycline, and °C, mixed with freshly prepared overnight culture, resistant to the remaining antibiotics. Similarly, poured on autoclaved Petri plates and allowed to amoxylin, ampicillin, penicillin G, trimethoprim and solidify under aseptic conditions. Whatman No. 1 sulfomethoxyzol inhibited the growth of E. faecium SHORT COMMUNICATIONS 557 (A and C) as indicated in (Table I). This finding is Table II.- Zone of inhibition of extracts of seeds and consistent with previous reports of non β-lactamase- green parts of Mamordica charantia against three strains of Enterococcus faecium and producing penicillin-resistant enterococci (Acar and Enterococcus faecalis. Buu-Hoi, 1988; Bush et al., 1989). E. E. faecium Table I.- Effect of antibiotics against three strains of Extracts faecalis Enterococcus faecium (A, B, C) and A B C D Enterococcus faecalis (D) Extracts of seeds E. Antibiotics used E. faecium N1 (n-Hexane) 0 0 0 0 faecalis against pathogens N2 (Chloroform) 0 0 0 0 A B C D N3 (Ethyl acetate) 0 20(+++) 0 0 N4 (Ethanol) 0 5(+) 0 6(+) Amoxylin (10 µg) R S R R N5 (Methanol) 20(+++) 0 0 0 Ampicillin (10 µg) R S R R Streptomycin(10 µg) R R S R Extracts of green parts Tobramycin (10 µg) R R S R N6 (n-Hexane) 0 0 22(+++) 0 Gentamycin (10 µg) S S S S N7 (Chloroform) 0 0 0 0 Ciprofloxacin (5 µg) S S S S N8 (Ethyl acetate) 6(+) 0 0 0 Pencillin G (10 µg) R R R R N9 (Ethanol) 26(+++) 0 0 8(+) Trimethoprim (5 µg) R R R R N10 (Methanol) 26(+++) 0 0 8(+) Sulfomethoxyzol (25 µg) R R R R Tetracycline (10 µg) S S S S Growth inhibition was recorded: very high (++++) 35-62 mm; high (+++) 21-34 mm; medium
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