STUDIES ON ANTIBACTERIAL EFFECT OF NIGELLA SATIVA ON E . COLI ISOLATED FROM HUMAN AND ANIMAL SOURCES

In this work a total of 47 E.coli isolates were tested and isolated from both human and animal sources. The oil extract of Nigella sativa was produced using cold mechanical compression method and used within 48 hours . The extract was applied to the isolated organism to test its inhibitory effect in comparison with varieties of standard antibiotics. Nigella sativa oil showed a good inhibition effect similar to most of the standard antibiotics in different concentrations, the higher the concentration of the oil the more marked the inhibitory effect . Out of 22 isolates of E.coli isolated from human 16 isolates (73%) of them showed inhibition with the oil extract in different concentrations. In 25 isolates of E.coli isolated from animal sources (diarrhetic calves) 16 isolates (64%) showed inhibition with the oil extract of Nigella sativa seeds in different concentrations. INTRODUCTION Multiple medicinal uses of Nigella sativa in the folk medicine encouraged many investigators to prepare various extracts of Nigella sativa, isolate active principles and to conduct experiments on laboratory animals and human beings using modern scientific techniques to demonstrate their pharmacological actions and therapeutic effects ( Badary and Gamal El-din, 2001). The discovery of antibiotics allowed the human beings to control microorganisms causing variety of diseases. Nowadays, drug resistance is a serious problem due to drug misuse by patients and physians and also due to addition of antibiotics to animal feeds as growth promoters, leading to the emergence of increasing number of antibiotic-resistant organisms (Franklin and Snow,1989)( Prescott et al., 1993)( Mckanne and Kandle, 1996). This serious problem may be overcome by seeking for new antimicrobial drugs of plant origin . Most importantly; both the oil and its active ingredients expressed antimicrobial and effects toward different microbes . Coupling these beneficial effects with its use in folk-medicine, Nigella sativa seed is 168 Enany et al., a promising source for active ingredients that would be with potential therapeutic modalities in different clinical settings. The efficacy of the active ingredients, however, should be measured by the nature of the disease. (Mohamed L.Salem, 2005) Aim of work The aim of this work is to detect the antibacterial effect of Nigella Sativa oil at different concentrations on E.coli bacteria isolated from both human and animal sources . MATERIALS & METHODS 1Plant material: Nigella Sativa (N.S) seeds were obtained from herbalist shops and were botanically identified and confirmed to be the seeds of that plant at Faculty of Science, Botany department, Seuz Canal University. 2-Bacterial isolates: Twenty two isolates of E.coli were isolated from human, (17 isolates from urine samples and 5 isolates from pus swabs). 25 isolates of E.coli were isolated from fecal samples of calves suffering from diarrhea. 3-Antibiotic sensitivity discs: Diagnostic antibiotic sensitivity discs were obtained from commercial sources. Table (1): Interpretation of zones of inhibition in agar diffusion method, for antibacterial susceptibility test: according to the NCCLS antibacterial susceptibility testing standard M2 – A7 and M7 – A5. Antibacterial agent Disc conc. Diameter of inhibition Resistant Intermediate Sensitive Tetracycline (TE) 30 μg 14 or less 15-18 19 or more Doxycyclin (DO) 30 μg 12 or less 13-15 16 or more Ofloxacin (OFX) 10 μg 12 or less 13-15 16 or more Antibacterial agent Disc conc. Diameter of inhibition Resistant Intermediate Sensitive Clindamycin (DA) 2 μg 12 or less 12-14 14 or more Chloramphenicol (C) 30 μg 12 or less 13-17 18 or more Gentamycin (GM) 10 μg 12 or less 13-14 15 or more Cefadroxil (CFR) 30 μg 14 or less 15-17 18 or more Amoxycillin (AMX) 30 μg 13 or less 14-16 17 or more Azactam (ATM) 30 μg 11 or less 12-13 14 or more Ciprofloxacin (CIP) 5 μg 15 or less 16-20 21 or more Kanamycin (K) 30 μg 13 or less 14-17 18 or more Norofloxacin (NOR) 10 μg 12 or less 13-16 17 or more Penicillin (P) 10 μg 19or less 20-27 28 or more SCVMJ, IVX (1) 2009 169 4Oil extraction from the seeds: The seeds were cold compressed mechanically. This cold method was necessary to reserve all the volatile oil during the compression of the seeds. The freshly extracted crude oil preserved in screw capped dark bottles to keep its effect for long time according to Published American literature and kept away from the high temperatures . The oil used as antibacterial during the first 48 hours only from the extraction. (Morsi , 2000). 5-Bacterial isolation :A loopful from the inoculated broth culture was streaked onto MacConky and Nutrient agar then incubated at 37 ْ C for 24 hours and examined for colony character according to Harvey and Price (1979). The pink colonies on the MacConkey agar indicate the presence of E.coli bacteria. 6Bacterial identification: * Microscopical examination: according to Cruikshank et al., (1975) . * Biochemical identification: Identification of the gram negative isolates was done according to Mitruka and Bonner (1976); Freeman(1985); Brooks et al.(1991); Pelczar et al. (1993); Johnson and Case (1995) and Collins et al (1995). The tests used in identification included: Methyl Red (MR), Voqus Proscour(VP), indole, and citrate utilization tests. E.coli is positive indole, positive M.R , negative V.P and negative citrate according to Cruikshank et al.,( 1975). 7-Preparation of oil extract of Nigella Sativa (N.S) as antibacterial sensitivity discs: The oil extract of the N.S seeds were dissolved in dimethoxy sulfoxide (DMSO) in a concentration of 80% , 60% , 40% and 20%. The solvent was also used as a negative control. The activity towards test organisms was evaluated using the diffusion plate method (Greenwood, 1983) (Hanafy & Hatem , 1991). : 8-Placement of antibiotic and oil antibacterial discs: With fine pointed burned forceps, the selected antibiotic were placed on the surface of the inoculated plates and pressed firmly into the agar to ensure complete contact with the agar. The discs were distributed evenly. Adequate spacing between different discs (at least 24 mm apart from center to center of every two discs) and between discs and the wall of the Petri-dish (at least 15mm away) was done. The discs which had the different concentrations of the oil extract , 100% , 80% , 60% , 40% , 20% and the disc which contain the negative control (DMSO which has no antibacterial effect) were placed onto the plates with the same previous method . (Hanafy & Hatem , 1991) and (Greenwood, 1983). After 24 hours incubation at 30-32 ̊C (this temperature suitable to preserve the volatile oil which present in the extract) the diameter of the bacterial inhibition zones which produced by the diffusion of antibiotic discs into the surrounding medium was measured using a transparent plastic ruler and compared with 170 Enany et al., the chart specific to each antibiotic type [Table (1)], These diameter readings gave the results of sensitivity or resistance to the antibiotics.The presence of inhibition of bacterial growth produced by diffusion of the antibacterial agent (different concentrations of the Nigella sativa oil extract) was determined by the appearance of the clear transparent inhibition zone or not. The degree of sensitivity is roughly estimated by the diameter of the inhibition zone considering the diameter of about 2.5 cm high (++++), of 2 cm moderate (+++) of 1cm mild (++) and less than 1 cm weak (+). RESULTS & DISCUSSION Twenty two isolates of E.coli were isolated from human (17 from urine and 5 from pus), while 25 isolates were isolated from feces of diharretic calves.As regarded to human E.coli isolates there was 16 of them were resistant to <=8 types of antibiotics and 6 were resistant to >8 types of antibiotics as shown in table (2). The 25 animal E.coli isolates showed 7 isolates resistant to <=8 types of antibiotics while 18 isolates showed resistant to >8 types of antibiotics as shown in table (3). The relation between bacterial response to antibiotics and inhibition of its growth by N.S oil at different concentrations are shown in the following tables . Table (2): The effect of Nigella sativa oil on the growth of Ecoli isolated from human source at different concentrations (n=22). Inhibition zone of bacterial growth at different concentrations of N.S oil 16 bacterial isolates resistance to<=8 antibiotics 6 bacterial isolates resistance to >8 antibiotics Total