Jafaar et al. Diyala Agricultural Sciences Journal, 8 ( 2 ) 28 – 34 ,2016 28 ANTIBACTERIAL PROPERTIES OF AQUEOUS GARLIC EXTRACT AGAINST SOME PATHOGENIC BACTERIA IN VITRO. Mohammed Mosa Jafaar Eman Sabaa Khamees Anaam Mahmood Abid * Food and Bio techniques Center- Agricultural Research Diroctorate Ministry of Science and Technology, P.O.Box,765,Baghdad- Mohammedmosa1@yahoo.com ABSTRACT The antibacterial effects of crust removed aqueous garlic extract using diethyl –ether against resistant pathogenic bacteria G+&G-isolates including Enteropathogenic E.coli 0.127, Bacillus cerues ,Listeria monocytogenes Salmonella typhi and Pseudomonas aeroginosa ,were studied .The first concentration don’t give any result(0.25,0.50.0.75) therefore increasing the concentration. Antibacterial activity by well-diffusion and used different concenteration 0.125% ,0.150% , 0.175 % .The result of inhibition zones of L . monocytogenic (33mm – 37mm) for gram positive while gram negative such as salmonella typhi don’t effect to the garlic extract. Key words: Garlic, Allicin, Antibacterial activity. INTRODUCTION Garlic is one of the edible plants, which has generated a lot of interest throughout human history as a medicinal panacea. A wide range of microorganisms including, bacteria, fungi, protozoa and viruses have been shown to be sensitive to crushed garlic preparations. Moreover, garlic has been reported to reduce blood lipids and anticancer effects. Chemical analyses of garlic cloves have revealed unusualconcentration of sulfur- containing compounds (1—3%) (Yeh and Liu , 2001.The compound turned out to be an oxygenated sulfur compound which they termed allicin,from the Latin name of the garlic plant,( Allium sativum).Pure allicin is a volatile molecule that is poorly miscible inaqueous solutions and which has the typical odor of freshly crushed garlic (Yin et al .,2002) proof of the chemical structure of allicin in 1947, when it was shown that allicin could be synthesized by mild oxidation of diallyl disulfide(Okochi et al. , 2000) The debate on the presence of allicin in crushed cloves versus its absence in odorless intact cloves was resolved after it was isolated (Akinyemi, 2000), identified, and synthesized an oxygenated sulfuramino acid that is present in large quantities in garlic cloves and which they named alliin. Alliin was found to be the stable precursor that is converted ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication 11/1/ 2015 . Accepted for publication 7/9/ 2015 . Jafaar et al. Diyala Agricultural Sciences Journal, 8 ( 2 ) 28 – 34 ,2016 29 to allicin by the action of an enzyme termed alliinase which is also present in the cloves(Tsao ,2001). Aim of the study Isolate and diagnose some types of pathogenic bacteria from various sources using the Vitek compact .The effect of the active ingredient of garlic (allicin) against pathogenic bacteria used in the study. MATERIALS AND METHODS Sources of test organisms and garlic: The microorganisms used for this study were isolated from stool sach as Salmonella typhi (Cowan , 1974) and Enteropathogenic E.coli 0.127 ( McNeish, 1992). Listeria monocytogenes from Cheeses (Beuchat and Ryu, 1997). Bacillus cereus from Of rice (Nygren, 1962), both of which caused diarrhea due to food poisoning. Pseudomonas aeruginosa from Infected wounds(Cowan, 1974).Occurent out food technical biology center / Microbiology Lab. Microbiological analysis of samples Samples were inoculated on tryptone soya broth (Oxoid, England), a general purpose broth that can support the growth of both aerobes and anaerobes.MacConkey agar used for the isolation of Enteropathgenic E.coli 0.127-like enteric organisms and confirmation by PCR (Higuchi and Dollinger , 1992), Xylosis lysine agar for isolation of Salmonella typhi and confimation by Antisera somatic O . Pseudomonas agar used for isolation of P.aeruginosa and confirmated by blue pigment . Frezer broth with supplement half frezer used for isolation L.monocytogenes . Bacillus selective agar for the isolation of Bacillus cereus and incubated at 37ºC for 24 hours. Primary isolates were repeatedly subcultured on fresh media using plate streaking techniques to obtain pure cultures. All strains are confirmated by Vitek compact-2 (biomerix). The purified isolates were stored in agar slant at 4ºC for later characterization and identification. Preparation of garlic extract Mixing 50 grams of garlic crust removed with 200 ml of distilled water and then nominated tow phases. The first raw canvas and the second of the nomination paper .Blending the filtrate with amount equal to the compound Diethyl ether in separating funnel for the purpose of Purifying the filtrate from Jafaar et al. Diyala Agricultural Sciences Journal, 8 ( 2 ) 28 – 34 ,2016 30 fatty oils done remove the bottom layer of the mixture and steamed in a rotary evaporator and the temperature of 50 C for the purpose of the filtrate purification filtrate from residues Diethylether .Filtrate stored in opaque botteles to use(Bonle and Desmukh, 1975). Antibacterial effects of raw garlic extract Hole diffusion technique using the stuck for each bacterial isolate by measured by McFarland device and planned to center muller Hintomarked.0.125,0.150.0.175 Mm of garlic extractions were added on surface center of bacteria medium used in the study. Figure 1. Gel electrophoresis of amplified bfpA (326bp), SLT I (894bp) and SLT II (478bp) from E. coli DNA isolated from stool specimens. Agarose (1.5%), 5 V/cm for 2 hrs, stained with ethidum bromide and visualized on a UV transilluminator. Lane 1 and 14 . 100 bp DNA ladder. Lane 2-5. Amplified bfp gene of Serogroups 55, 86, 127 and control respectively. Jafaar et al. Diyala Agricultural Sciences Journal, 8 ( 2 ) 28 – 34 ,2016 31 RESULTS AND DISCUSSION Table 1: Sensitivity test for Garlic extract by Diethyl ether as antimicrobial for some Pathogenic bacteria as MIC (minimum inhibitery concentration) . contro l L.monocytogenes (mm) Pathogenic E.coli 0.127 (mm) B.cereus (mm) P.aeruginos a (mm) S.typhi (mm) Concentra- tion of garlic extract ( mm) Zero - - - - - 0.25 Zero - - - - - 0.50 Zero - - - - - 0.75 Table2: Sensitivity test for Garlic extract by Diethylether as antimicrobial for somePathogenic bacteria as MIC (minimum inhibitery concentration) control L.monocytogen es (mm) Pathogenic E.coli 0.127 (mm) B.cereus (mm) P.aeruginos a (mm) S.typhi (mm) Concentra- tion of garlic extract (mm) Zero 33 11 17 - - 0.125 Zero 37 13 19 - - 0.150 Zero 37 17 19 - - 0.175 Show Table1:the garlic extraction for first concentration didn , t give any result therefore the concentration increasing. Bacterial isolates from the different samples were principally Enteropathogenic coli 0.127 ,B. cereus,and L.monocytogenes, were used for sensitivity test with garlic concentration table 2, which shows the zones of inhibition of bacterial growth by garlic,L.monocytogenes was highest sensitivity to garlic extract, B.cereus was least sensitive against garlic extract ,and the Enteropathgenic E.coli 0.127 Less sensitivity. While P.aeruginosa and S.typhi not affected garlic extract all the concentrations used in the study. the active ingredient of garlic the compound turned out to be an oxygenated sulfur molecule which they termed( Allicin), (Hughes and Lawson, 1991), acts by partially inhibiting DNA and protein synthesis and also totally inhibiting RNA synthesis as a primary target (Feldberg et al, 1988). and thus interferes with DNA transcription and other activities involving DNA (Prescott et al, 2005). In this study gram-negative bacterial isolates were less sensitive to garlic than gram positive. The cell wall structural nature of gram-negative enteric bacteria may be responsible for the susceptibility resisting. For instance the bactericidal effect of garlic might be due to the structural characteristics of organismswhich Jafaar et al. Diyala Agricultural Sciences Journal, 8 ( 2 ) 28 – 34 ,2016 32 play a role in the bacterial susceptibility to garlic constituents (Tynecka and Gos, 1975), particularly Gram positive contains only 2% lipid (Salton, 1964) so that the lipid content of the membranes will have an effect on the permeability of allicin and other garlic constituents. Hence these phenomenons may favor the destruction of the cell wall and genetic materials of gram positive The lipid composition of cell wall has been found to have an influence on the permeability of hydrophobic and volatile bioactive substancesin garlic (White , 1998) whereas the cell wall of gram-negative bacteria contains 15-20% polysaccharides and 10-20% lipid(Carpenter,1968). In a study of (Sivam,1998) in which he The cell membrane of E. coli has been reported to contain 20% lipid . The polysaccharides and the lipid contents of the cell wall affect of very high the permeability of allicin and other garlic constituents, the effect of garlic extract is most pronounced on enteric bacterial pathogens.The absence of resistance to garlic enhances its ability to effectively act against even highly resistant bacterial isolates, such as Pseudomonas aeruginosa. It therefore appears attractive that garlic affect DNA and RNA syntheses . In another study this may be responsible for the difference in susceptibility to garlic between gram negative Helicobacter pylori and gram-positive Staphylococcus aureus (Cellini et al., 1996) . CONCLUSION Garlic (A. sativum) has antimicrobial properties against gram positive It has both a bacteriostatic and bactericidal activity when tested in vitro using crude preparation of garlic. Therefore, garlic may be used successfully for treating food poisoning causative agent like gram positive further in vivo studies are necessary. REFERENCES Akinyemi K.L. 2000: Prevalence of multiple drug resistance Salmonella typhi among clinically diagnosed typhoid fever patients inLagos, Nigeria. Zatur-forsch;55:489–493. Beuchat , L.and J.Ryu .1997.Produce handling and processing practices .Emerg. Infect. DiS 3: 459 465 . Cellini L.,E.D.Campli,M.Masuli,S.D.Bartolomeo and N. Allocati 1996. hibition of Helico- bacter pylori by garlic extract (Allium sativum). FEMS Immunol Med. Microbiol.; 13: 273-7. 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Diyala Agricultural Sciences Journal, 8 ( 2 ) 28 – 34 ,2016 34 خواص المضاد المايكروبي للمستخلص المائي للثوم ضذ بعض انواع البكتريا الممرضه خارج الجسن الحي انعام محمود عبد ايمان سبع خميس محمد موسى جعفر وزارة العلوم والتكنولوجيا -دائرة البحوث الزراعيه -االحيائيه التقانات الغذائية*مركز المستخلص ثير المسمملص ا المممبلث ل اممىا المممساق رهمما الداممرا بسمملصياا داض اثيمم ا اممر م ممبد أتمم ةسممادرتمم وهمث ل م مبداة البكلير مة ا والمىجبما لبمب ة مراا خمي مسمة ممسية ررخمية ردبورمةل بكلر ب السمبلب E.coli 0.127 ,Bacillus cereus ,Listeria monocytogenes , Salmonella typhi و Pseudomonas aeruginosa 0.25%فممث البيا ممة اسمملصيري ترا يممس هيممر رمم ثرا اليراسممة قيممي خمميالمسمملص ا اظهممرة الهلممبلج اممب ل ز ممبدا اللرا يممس وفمم لج,ولمم تي ممث تض لممب %0.75,%0.50, 0.25ا رصل فترا يس راا بسلصياا اي لابر فث الحفر حيث اسلصيري البكلر ب المىجبة والسبلبة لبب ة -ر م Listeria monocytogenes (33% و ب ي لبلج اللابيط ل بكلر ب المىجبة ,0.175 0.150 ,% ت الكاف مم اليمسية البكلير ما لر ب السبلبة را السبلمى يال ل تلأثر مسلص ا الاىا ر ( يهمب البك33 ( PCRوالفحىصبة الجس ئيا ) API 20 E بسلصياا الفحىصبة المىرفىلىجيا والكيمىحيى ا را سي , فيبلية الم بد الحيىض لالاىا , اي الكلمات المفتاحية :