مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Turbidimetric Method for Measuring of Antibacterial Effect of 2-Methyl-4-(3,4 dihydroxy Phenyl)-Thiazole on the Growth Activity of Escherichia Coli and Pseudomonas Aeruginosa A.K. Abbas Department of Physiological Chemistry, College of Medicine. University of Baghdad Received in : 26 April 2009 Accepted in : 18 August 2009 Abstract Thiazoles are heterocyclic nitrogenous compounds, which were given a great attention due to their antibacterial effect, antihypertensive, stimulating glucose absorption (like antibiotics), acts as cytoprotective agent. The present work is conducted to evaluate the degree of antibacterial activity on the growth activity of Escher ichia Coli (E.Col i) and Pseudomonas aeruginosa (P. aeruginosa) by incubation with 2-methyl-4-(3,4 dihydroxy phenyl)-Thiazole compound (MDHPT). Optical density (O.B) was measured by turbidmetric method, any increase in O.D would represent the increase in the bacterial growth .The results showed that there was a 75.7 % inhibition when using 190.4 g/ml (MDHPT) solution and 64.3% was obtained when using concentration of 369 g/ml. It has been concluded that (MDHPT) compound shows a bacterial biological activity. Key words: Thiazoles, E.Col i, P . aeru ginosa, i n c u b a t i o n m e d i u m , incubator, minimum inhibitory concentration (MIC) Introduction The study of new derivative of Thiazole has been given a great attention, due to their antibacterial activity [1, 2, 3] antihypertensive [4] and stimulation of glucose absorption like antibiotics [5], lowering cholesterol [2], shows bacteria static activity [3] and acts as cytoprotective agent by induction of glutathione-S-transferases (GST) represent not only cell detoxification and survival but also cancer prevention [6,7]. Several attempts were carried out to elucidate the effect of different antibiotics related to this compound on the growth activity of pathogenic St ap hy lococcus aureus [2,3,8,9,10,11,12,13,14] This work is related to the action of 2-Methyl-4-(3,4 dihydroxyphenyl)-thiazole (MDHPT) on the growth activity of Escherichi a Coli (E.Col i) and Pseudomonas ae ru gi nosa (P. ae ru gi nosa ) either in solid or in liquid media . مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Both bacteria are of normal flora of the intestine and they cause different diseases especially in weak people [15]. The clinical records revealed that 83% of patients with leukemia having infection with Pseudomonas aeruginosa [16].This bacteria is also resistant to many antibiotics which may be attributed to alteration in cell membrane transport system. [17]. The aim of this study is to evaluate the degree of the antibacterial inhibitory effect of 2- Methyl-4-(3,4 dihydroxyphenyl)-Thiazole on the growth activity of E.Coli and P .aeruginosa and to find the effective dose required . Materials and Methods 2-Methyl-4-(3,4 dihydroxyphenyl)-Thiazole hydrochloride, was synthesized by professor Shukri, J, Department of chemistry, College of Science , University of Baghdad [1]. (2-Methyf-4-(3,4 dihydroxyphenyl)-Thiazole) A stock solution was prepared by dissolving (4 x 103 g/ml) in water with few drops of glycerol. To prepare a starting culture of bacteria, a loop full of stock culture from each type of bacteria was added to 50 ml of tryptose phosphate broth medium (29.5 g/liter, pH 7.3) and incubated in shaking water bath for 20-24 hours at 37° C and at 200-250 rpm (rotation / minute ). Experimental flasks of 250 ml with side arm, each contains 20 ml of sterilized culture medium. Each flask was inoculated with 1.0 % (V/V) of both used bacteria suspension which contain 2.4 x 107 cell / ml [18]. The number of bacterial cells was calculated by using Neuber heamocytometer [19]. (MDHPT) solution was added to the inoculated media at the beginning of cultivation, the flasks were closed with cotton plugs under sterilized conditions. A control was run under the same conditions without the addition of thiazole solution. The flasks were put in the shaking incubator at 200-250 rpm to ensure adequate aeration at 37 o C for 24 hours. Changes in the concentration of viable bacteria during lag, exponential and stationary phases were measured by changes in the optical density at 607 nm, using EEL colorimeter (Evans Electroselenium LTD. Halstead, Essex, England). مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Results Different concentrations of 2-Methyl-4- (3,4- dihydroxyphenyl)-thiazole ranging from (19.9 g/ml (8.1 x 10 -2 ) - 190.4 g/ml (7.8 x l0 -1 M)) were added to E. Coli culture media (after six hours of incubation). The results showed an inhibition of (24.3, 37.8, 48.6 and 75.7 %) in the growth rate of the bacterial culture, at concentrations of (19.9 g/ml (8.1-2 M), 59.1 g/ml (2.4 x 10-1 M), 97.5 g/ml (4 x 10-1 M), and 190.4 g/ml (7.8 x 10-1 M )) respectively; Table 1, Fig. l. Inoculation with P. aeruginosa required higher (MDHPT) concentrations than that used with E. Coli at the same conditions. Concentrations of (135.2 g/ml (5.5 x 10-1 M), 244.4 g/ml (l.0 M), 313.4 g/ml (1.28 M) and 369.6 g/ml (1.49 M)) showed an inhibitory effect of (16.13, 38.7, 48.39, and 64.3%) respectively in comparison to control activity for E.Coli and P. aeruginosa . At the same experimental conditions Table 2, Fig 2. The results were the mean of ten experiments with P< 0.05. All data are expressed as a mean of ten experiments with P< 0.05. Discuss ion (MDHPT) was studied with St ap hy lococus aureus results show bacteria static activity of 70% inhibition (MIC 200 g/ml = 0.82 M) [2,3]. In this study a bacteria static activity of 75.7 % was achieved with a concentration of, 190.4 g/ml (8.1uM) inoculated, with E.Coli in liquid medium after 6 hours of incubation time. This results were inconsistent with what was reported by (Faraid, Al-Chalabi et al, and Shukri, J. et al), while a higher concentration solution of (MDHPT) 313.4 g/ml (l28 M), was required to achieve nearly the same rate of inhibition (64.3 %) as with E. Coli [2, 3 ]. Bacteria static activity was achieved, this may be explained by the presence of potent group(s) that achieved the inhibitory action on the growth activities of each of gram- negative E.Coli and P.aeruginosa , and also gram-positive Staphyloco ccus aureus (St. aureus ). The presence of active Methyl group (-CH3) at position 2 of the Thiazole ring which can react with carbonyl groups of the peptidoglycan or lipopolysaccharide (LPS) of the bacterial cell membrane [2, 20, 21]. The dissociation of phenolic groups present in the compound (MDHPT) may cause a release of excess protons (H + ) to the medium which may be picked up by the (-NH2) of the peptidoglycan of the bacterial cell membrane forming excess (-NH + 3) which may create an ionic attraction leading to a cell wall damage[3,19]. The cell wall of gram-negative bacteria has distinctly layered appearance, the inner region consists of monolayer of peptidoglycan, while the outer cell wall bilayer inward facing lipids are macromolecules called lipopolysaccharides (LPS), half the mass of the outer membrane consists of Braun proteins which covalently linked to peptidoglycan layer, adjacent outer lipopolysaccharides are held together by electrostatic interaction with divalent ions ( Zn +2, Ca+2, Mg+2), while gram-positive cell wall is multi-layered network which appears to be continually-growing by the addition of new peptidoglycan linear hetropolysaccharide chain cross-linked by short polypeptides at the interface with concomitant outer surface .[21,22,23]. There may be another possibility to explain the inhibitory effect it-may-be that the activity of (MDHPT) is due to its ability to chelate essential metal ions e.g. (Zn, Mg +2 ) from bacteria which cause a disturb of these interaction of gram-negative (E.Co li & P.aerugenosa) by binding to the divalent ions in the cell wall which .cause the disintegration. Also chelation may occur to those ions that required for enzymes activation مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 processes occurred in bacterial cell wall. [ 20,21,22]. Variation in the results obtained with both bacteria under study could be due to high variation in amounts of the peptidoglycan present in the gram-negative or gram positive bacteria cell wall membrane [21,24]. References 1. Shukri, J. (1977) Brenzcatechin substituierte, Thiazofe, Wiss, z. Univ. Halle, xxVI, 77M,H,5(S): 59-61. 2. Al-Chalabi, F.A.J.; Elham, M.H.; Jaafar and Aziz, HH, Al-Lami, (1984) Effect of new thiazole derivatives on the growth of staphylococcus aureus, J.B.C. Vol. 15 (2). 3. Shukri, J, Shubber, A; Al-Chalabi, FAJ, Khalifa, A and Hassan, F. (1979) New thiazole compounds as potential antibacterial agents. Iraqi J. Sci., 20 (l):75-86. 4. Smith-Klein and French Laboratories, (1972) Phil. USA, Private report. 5. Al-Lami, AHH; Al-Chalabi, FAJ, and Jaafar, MH, (1983) The influence of new thiazole compounds on glucose absorption from isolated intestinal segments of the rabbits. J. Biol. Sc. 214(1): 3-14. 6. Sang Geon Kim and Seung Jin Lee, (2007) Toxicological Sciences, 96 (2) 206-213; doi: 10, 1093 Tox Sci/K 1175. 7. Kim, H.P. Ryter, S.W., and Chei, A,M ., 2005; Annu.Rev. Pharmacol. Toxicol. 8. Collins, J.F. (1974) Application of Prokaryotic Genetics in Biochemical Studies, in Companion to Biochemistry Bull E, Lagnado JR, Thomas JO, and Tripton KF; pp.471, Longman, London. 9. Eagle, H. and Misselman, A.D.(1948) The rate of bactericidal action of pencillin in vitro as a function of its concentration and its paradoxically reduced activity at high concentrations against certain organisms.J. Exptl. Med. 88:99-131. 10. Mayhall, M. and Apollo, E. (1980) Effect of storage and changes in bacterial growth phase and antibiotics concentrations on antimicrobial tolerance in staphylococcus aureus,Antimicrobial-agents chemother., 18(5):784-788. 11. Peterson, E.; Fairahter, Morrison, J. and Cesario, T. (1981) Effect of the herbicide paraquate dichloride on bacterial of human origin. Appl. Environ. Microbial: 41(l): 327-328. 12. Raynor, RH; Scott, DF, and Best, Co.K. (1976) Oxicillin-Induced lysis of staphylococcus aureus.Antimicrobial. Agents Chemother. 16:134-140. 13. Watnakunakron, C. and Cuerriero, J.C.(1981) Interaction between vancomycin and rifampin against staphylococcus aureus. Antimicrobial. Agents Chemother.: 19(6): 1089-1091. 14. Jawad, A.M. and Jaffer, H.J. (2008) In vitro antimicrobial activity of total sequiter penlactones and phenols isolated from some Iraqi plants. Um salama. Science Journal, 5(1): 80-83. 15. Ernest Jawetz, Melnick, JL.Adelberg, EA (1974) Review of medical microbiology, 14 th Ed., Lange medical publication Canada; p. 211-212. 16. Micheal, V. Viola(1967)Acute Leukemia and Infection. J.A.M., 201-203. 17. Spicar, A.B.(1976) Increased antibiotic sensitivity in pseudomonas aeruginosa following passage in carbenicillin containing media. J. App. Microbial., 40: 33-45. 18. Jaafar, E.M.H.(1976) A study on drug resistance Saccaromyces Cervisiae M.Sc. Thesis, Warwick University England. 19. Cruickshank, R, Duguid, JP, M armion, BLA, Medical Microbiology, Vol. 2, 12th Ed. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 20. Pamela,C. Champe and Richard A Harvey "Lippincott's Illustrated Reviews: Biochemistry, 2nd ed. Copyright (1994) J.B. Lippincott Company. 21. Michaels Blader, Spring. (1998) BCH 5425 Molecular Biology and Biotechnology 16:925-928. 22. Al-Chalabi, F.A.; Al-Najjar, F.A.; Al-Ani, S.M. The effect of some metal ions and their chelates on the viability of staphylococcus aureus J Ibn Al-Haitham, M Pure and App Sci., Vol. 5(1): 23. Al-Chalabi, F.A.; Mehdi, W.K. and Al-Karaghouli, K.(2003) The effect of Nickel celates on the reactivation of inhibited Staphylococcus. 24. Huang, J.W.; Blaylock, M.J.; Kapulnik, X and Ensley, B.D. (1998) Environ., Sci. Technol. 32:2004-2008. Table (1): Represents counts of viable E.Coli cells and % inhibition when 2-Methyl-4- (3,4 dihydroxyphenyl) -Thiazole compound was added to inoculation medium compared to control. Mean of ten experiments P< 0.05 Time hours Test No. of viable E.Col i Cells Added 1.99 g/ml (0.8 M) (MDHPT) % Inhibition Control 0 2.4 x 107 0.0 2.4 x 107 2 2.6 x 107 24.3 3.6 x 107 4 7.2 x 108 32.8 3.6 x 109 6 3.5 x 10 9 48.6 8.8 x 10 9 24 4.9 x 10 12 75.7 7.0 x 10 12 مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Table (2): Shows counts of viable P. aeruginosa cells and % inhibition when 2-Methyl-4- (3,4 dihydroxyphenyl)-Thiazole compound was added to inoculation medium, compared to control. Mean of ten experiments P< 0.05 Time hours Test No. of viable P. aeruginosa Cells Added 369.6 g/ml (1.49 M) (MDHPT) % Inhibition Control 0 2.4 x 107 0.0 2.4 x 107 2 1.5 x 108 16.13 8.6 x 107 4 1.3 x 109 38.7 8,0 x 109 6 3.9x 1010 48.39 8.0 x 1010 24 5.8x 10 11 64.3 9.0x 10 11 Fig. (1): Effect of various concentrations of 2-Methyl-4 3,4 dihydroxyphenyl) 4- Thiazole hydrochloride on the growth activity of E. Coli a- Control bacterial growth of E.Coli, b-Inoculation medium contains 8.1 x 10 -2 uM of (MDHPT). c- contains 2.4 x 10 -1 M (MDHPT), d- (MDHPT) 4 x 10 -1 M e- (MDHPT) 7.8 x x 10 -1. Experimental conditions were described in the method. Data are expressed as a mean of ten experiments , P<0.05, مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Fig. (2):Effect of various concentrations of 2-Methyl-4- (3,4 dihydroxyphenyl) - Thiazole hydrochloride (MDHPT) on the growth activity of P. aeruginosa a-Control bacterial growth, b- (MDHPT) 5.5 x 10 - 1  M. c- (MDHPT) 1.0 M. d- (MDHPT) 1.2 M e- (MDHPT) 1.49 M. Experimental conditions as in Fi g l . P< 0.05. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 مستوى النمو للبكتریا فيتأثیر مضادات البكتریا لمركبات الثایوزول )Pseudomonas aeruginosa و Escherichia coli( احسان خلیل عباس قسم الكیمیاء ، كلیة الطب ،جامعة بغداد 2009 آب 18: قبل البحث في2009نیسان 26: استلم البحث في الخالصة ت تعد مركبات الثایوزول العضویة من المركبات التي أعطیت أهمیة كبیرة في ضوء خصائصها بوصفها مضادا ع ًبكتیریة وحیویة فضال عن كونها محضرات لعملیة امتصاص سكر الكلوكوز وتقدیر فعالیة التثبیط لنمو البكتریا لألنوا )Pseudomonas aeruginosa و Escherichia coli ( باستعمال مركبات الثایوزول العضویة حاضنات فقد استعمل .لهذا الغرض) MDHPT( ثایوزول –) ثنائي هیدروكسي فنیل3,4 (– 4 –المركب العضوي ثنائي المثیل . قیاس الكثافة الضوئیة تعني ازدیاد تثبیط نمو البكتریا التي تم اختیارها في هذه الدراسةأظهرت النتائج من خالل بینما كانت النسبة %) 75.5(لها فعالیة تثبیط بنسبة ) g/ml 190.4(كما أثبتت النتائج بأن تركیز مادة الثایوزول ًان له تأثیرا كبیرا ) MDHPT(تي عد المركب وفي ضوء النتائج ال) g/ml 369(عند التركیز %) 64.3(المثبطة للنمو ً .في نمو البكتریا )MIC(، حاضن ، أقل تركیز تثبیطي )حاضن(مركب الثیازول، وسط حراري : الكلمات المفتاحیة مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012