IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.23 (3 ) 2010 Role of Omeprazole in Enhancement of Antibiotic Resistance in E. coli S. A. Al-Bak ri, R. R. Jabri, E. A. Ajeel Departme nt of Biotechnology, School of Applied Science, Unive rsity of Technology. Abstract E. coli was isol ated, and it was Gram-negative rod bacteria that was colony circular, regu lar edged, thick somewhat glitter and viscous(less). It was lactose fermenter bacteria and belon gs t he family of Enterobacteriaceae. E. coli showed sensitivity to all used antibiotics except Ery thromy cin (E), Cloxacellin (CX), Rifamp in (RA), Cephalothin (KF), Amp icillin (AM ), and Penicillin (P). The exp erimental r esults of antibiotic sensitivity of E. coli in media containin g different concentrations of omep razole, a p roton p ump inhibitor, showed an enhancement of resist ance by decreasing the sensitivity of E. coli inverse ly with drug concentration against the antibiotics that E. coli was sensitive to. It seems t hat omep razole changed cell membran e potential of E. coli which led to depolarization of cell membran e as a result of inhibition of the proton p ump mechanism. This made the bacterial cell not willin g to uptake antibiotics. Introduction Escherichia coli is a member of the genus Escherichia that includes in the family of Enterobacteriaceae [1]. It is a Gram-negative, nonsp oring, facultative rod that ferments lactose with gas formation within 48 hour, at 35°C [2]. E. coli is the best st udied bacterium and the exp erimental organism of choice for many microbiologists. It is a major inhabitant of the colon of human and other worm-blooded animals[3,4]. It is the major causative agent of urinary tract infection (UT I). UT I is treated by antimicrobial drugs that destroy p athogenic microorganisms at low concentration called M inimum Lethal Concentration (M LC) or inhibit their growt h at low concentration called M inimum Inhibitory Concentration (M IC) [5,6]. Over the p ast several y ears, the medical community has become incr easin gly concerned over the ability of certain bacteria to dev elop resistance to antibiotics [7,8]. Accordingly , there is a dan ger of losing the batt le against certain p athogens (disease causin g or ganisms) by using the antibiotics in the treatment[7]. Bacteria do not beco me resistant to antibiotics without p resence of mechanisms by which resistance may be conferred [9,10]. There are four main mechan isms by which microorganisms can exhibit resistance to antimicrobials which: dru g inactivation or modification, alteration of target site, alteration of metabolic p athway, or reducing dru g accumulation [7]. Furt hermore, there are several means throu gh which the uptake of a dru g into a cell can be reduced: changes in the st ructure of the cell membrane, loss, or mutations of p orins in the cell membranes, and active efflu x of the drug fro m the cell [11,12]. The drug efflu x sy stems are membrane transp ort p roteins. Proton motive force (p mf) system is one of the drug efflux mechanisms, is p rincipally found in bacteria and yeasts. T he p mf is also known as the electrochemical p roton gradient, and a chemical p roton gradient. It functions as antip orter, and therefore mediates dru g efflu x in exchange with p roton translocation into t he cell [13]. Current study dealt with the role of proton motive force mechanism in dru g efflu x p ump in E.coli, a b acterial model, by using omep razole, an inhibitor of p mf mechan ism. Omeprazole is one of the most widely p rescribed drugs internationally and is used in the IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.23 (3 ) 2010 treatment of dy sp ep sia, p ep tic ulcer disease (PUD), gastroesop hageal reflu x disease (GORD/GERD) and Zollinger-Ellison sy ndrome [14]. Materials and Methods Isol ation of E. coli: An E. coli was isolated from p atient with UTI, cutivated in M acconkey agar (HIM EDIA) p late, and then cultivated in Nut rient agar (HIM EDIA), and then its shap e was identified [15]. Anti biotic Se nsitivity (Disk Diffusion Method): Ant ibiotic sensitivity for isolated bacteria was don e by using a combination of antibiotics d isks (Bioanalyse); including Cloxacellin (CX) 1μg, Erythromy cin (E) 15 μg, Tetracycline (TE) 30 μg, Amp icillin (AM ) 10μg, Gentamicin (CN) 10 μg, Na lidixic Ac id (NA) 30μ g, Cephalothin (KF) 30μ g, Neomycin (N) 30μg, Penicillin (P) 10 μg, Lincomycin (L) 2μg, Cephotaxime (CTX) 30μg, Trimethop rime 1.25μg + Sulfamethorazole 23.75 μg ( SXT ) 25 μg, Chloramp henicol (C) 30 μg, Nitrofurantion (F) 300μg, Rifamp in (RA) 5μg, and Tobramy cin (TOB) 10μg. These disks were st ored at 4°C using disk diffusion method as the following: 0.1ml of each st rain was cultured by sp reading on the suface of nutrient agar p late, and then antibiotic d isks were p laced on the surface of agar using st erile forceps and incub ated at 37°C for 24 hr. The results were indicated according to formation of inhibition zone around the disk for sensitive or not formation of inhibition zone around the disk for resistance. [16]. Se nsi tivity to Omeprazole: Two different concentrations of omeprazole (stock solution of omep razole of 1 mg/ml. was p repared by dissolving the gr anules of 1 cap sule [20mg] of omep razole [Ajanta] in 20ml of distilled water and st ored at -20°C) were p repared (100 and 300 μg/ml), and each concentration was added to 20ml of Nut rient agar and left to solidify , and then E. coli was cultivated by streaking, and left to incubate at 37°C for 24 hr. Then the sensitivity of E.coli to omep razole different concentrations was detected. [17,18]. Testing the Role of Omeprazole in Anti biotic Se nsitivity: Each of the p repared concentrations of omep razole (100 and 300 μg/ml) was added to 20 ml of Nut rient agar and left to solidify . E. coli was inoculated on the surface of each p lates of Nutrieint agar (the p lates that contain omep razole). The antibiotic disks (same antibiotic disks that were used in antibiotic sensitivity test) were added to t he surface of Nutrient agar of each p late. Plates were incubated at 37°C for 24 hour. The results were detected and recorded according to formation, absence, in creasing, or d ecreasing of inhib ition zone around the disk .[17,18]. Results and Discussion Isol ation of E. coli : E. coli was p urified by cultivation on M acConkey agar med ia, the morphological characteristics and p rop erties of E. coli wer e determined as: cir cular, regular edge, thick somewhat, glitt er pink, viscous, and lactose fermenter [2]. Anti biotic S ensitivity: The sensitivity to antibiotics was determined and the results indicated that E. coli was sensitive to 10 antibiotics and resistant t o 6 antibiotics from 16 typ es of antibiotics used (Table 1). These results were used as a control for further comp arison (Table 3). Se nsi tivity to Omeprazole: The recommended dosa ges for p atients taking omep razole are 10mg, 20mg, or 40mg. But the most frequent side effects of omep razole are headache, diarrhea, abdominal p ain, nausea, dizz iness, trouble awakening and sleep deprivation [19]. Omeprazole may be associated with a greater risk of hip fractures [20], Clostridium difficile diarrhea, and heart p roblems, including card iac arrest [21]. The study dealt with minimum concentrations (100, and 300 μg/ml) (M IC) of omep razole to minimize its side effect on human and ensure its activity on E. coli. (17,18). E. coli was cultivated in each concentration of omep razole containing agars after 24 hr. of incubation at 37°C. The st udy found that E. coli grew at both omep razole concentrations (Table 2) and this drug showed no killin g effect on E. coli [22,23]. For t his reason t his IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.23 (3 ) 2010 research used omep razole to find out its role in modu lation of antibiotic resistance in E. coli. Since omep razole is a p roton p ump inhibitor [13,22]. Omeprazole - Anti biotic Sensi tivity Test: Results in (Table 3) illustrate the antibiotic sensitivity of E. coli in petridishs containing two concentrations of omeprazole; 100 μg/m and 300 μg/ml. There wer e obvious decreasing in the diameters of inhibition zones of the most antibiotic typ es that E. coli was sensitive to reverse ly with omep razole concentrations as comp ared with control group in (Table 3). In addition, E. coli exhibited its resistance against TE and F at 300μg/ml of omep razole. Accordin gly, the tot al antibiotic types that E. coli was resistant t o, had increased at 300μg/ml of omeprazole containing medium to become 8 fro m 16 antibiotic typ es (Table 3). These results p rop osed that omep razole had an imp ortant role in the enhancement of resistance of E. coli against antibiotics [24]. Omeprazole is a p roton p ump inhibitor [13,22]. The proton p ump sy stem is an integral membran e p rotein that is cap able of grabbin g p rotons from the matrix (the sp ace enclosed by the two membranes) and releasing the protons into the inter-membran e sp ace. The confined p rotons create a difference or gr adient in both p H and electric charge and establish an electrochemical p otential. Because there's a high er concentration of p rotons in the inter-membran e sp ace compared to inside the cell, there's p ressure to return p rotons down the concentration gradient to restore the balance. This p ressure is called the p roton motive force (p mf) [25]. In current st udy , after treatment of E. coli with omeprazole, a gradient in electric char ge might be created, the concentration of p rotons inside the bacterial cell was higher than its concentration in the inter-membran e sp ace [26]. There was no way to restore the balance by p ump ing the p rotons outside the cell [27] due to the action of omep razole as a p roton p ump inhibitor. Blocking the p assage of p roton p umping seemed to create a decr easin g in cellular membran e potential (Dep olarization of cell membran e) [28]. Depolarization of cell membrane may make E. coli not willing to up take the antibiotics into the cell [29]The results of antibiotic sensitivity in omep razole containing media give an indir ect suggestion that there were no effect of gradient in pH [30] on t he downsizing of p orins to make the p ermeability of cell membrane se lectable [31] for the antibiotics of low molecular we ight [32]. This suggestion depends on that TE and F don’t have larger molecular weight than the other antibiotics (Table 4) to let E. coli showed resist ance or decreasing the sensitivity against them after the treatment with 100 μg/ml omep razole as found in (Table 3), while the antibiotic sensitivity has been decreased against the other types of antibiotics at 100 μg/ml and 300 μg/ml o mep razole containing media. These results are in agreement with the st udy of Perlin, and his colleagu es [24]. They reported the electrogenic behav ior of p roton transp ort by the H + -ATPase in Saccharomyces cerevisiae. H + -AT Pase is encoded by pma1 gene. The study found that mutations within pma1 may alter st eady -st ate membran e p otential formation, p ossibly through a chan ge in the electro genicity of the H + -ATPase. This make the mutant exh ibited its resistance to hy gromycin B that may be mediated via depolarization of the cellu lar membrane potential. Conclusion The exp erimental results revealed that the treatment of E. coli with omep razole, a p roton p ump inhibitor, did not modulate the resistance phenomenon in E. coli but it enhanced the resistance by decreasing the sensitivity of E. coli inversely with the drug concentration against the antibiotics t hat E. coli was sensitive to (before the treatment with omep razole). The study suggested that the decreasing in the sensitivity may mediate by depolarization of cellu lar membrane v ia a p otential change in cell membrane as a result of p roton p ump inhibition by omep razole. Accordin gly, the modulation of antibiotic r esistance by a p roton p ump inhibitor is not recommended because the treatment of bacterial infection may become more comp licated. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Re ferences 1. Ewing, W.H. (1986). “Edwards and Ewing's Identification of Enterobacteriacea e”, 4th ed. Elsevier, New York. 2. Feng, P., Weagant, S.and Grant, M . (2002). “Enumeration of Escherichia coli and the Coliform Bacteria”. Bacteriolog ical Analytical Manual (8th ed.). FDA/Center for Food Safety & App lied Nutrition. 3. M adigan M Tand M artinko JM (2006). “Brock Biology of M icroorganisms”, 11th ed., Pearson 4. Conway , P.L. (1995). “M icrobial Ecology of the Human Large Intestine”. In: G.R. Gibson and G.T . M acfarlane, eds. p .1-24. Human colonic bacteria: ro le in nutrition, p hy siology , and pathology . CRC Press, Boca Raton, FL. 5. Andrews, J. M . (2001). “Determination of M inimum Inhibitory Concentrations”. 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(1978). “M olecular mechanisms for p roton transp ort in membran es”. Proc. Natl. Acad. S ci. USA. 75:298-302. 27. DeCoursey , T.E. (2003). “Voltage-gated p roton channels and other p roton transfer p athways”. Physiol. Rev. 83:475-579. 28. Thomas, R.C., and R.W. M eech. (1982). “Hy drogen ion currents and intracellu lar p H in depolarized voltage- clamped snail neurons”. Nature. 299 :826-828. 29. Erdogan, Seref ; Oz gunen, Ker em Tuncay ; Oz gunen, Tuncay . (2004). “Effects of H+-K+ AT Pase Inhibitors (Omeprazole and Lansop razole) on Fertilization-Induced Bioelectrical Pot ential Changes in the Egg of the Fro g, Rana cameranoi”. Turk J. Vet. Anim. Sci. 28 : 329-336. 30. Demaurex, N., Grinst ein, S., Jaconi M ., Schlegel, Lew,W. D.P. and K. H. Krause. (1993). “Proton currents in human granulo cytes: regulation by membrane p otential and intracellular p H”. J. Physiol. (Lond.). 466:329-344. 31. Todt, Jell C., Rocque, Warr en J., and M cGroarty , Estelle J. (1992). “Effects of p H on Bacterial Porin Function”. Biochemistry. 31: 10471-10478. 32. Danilchank a, Olga; Pavlenok, M ikhail;and Niederweis, M ichael (2008). “Role of Porins fo r Upt ake of Antibiotics by Mycobacterium stnepnatik”. Ant imicrobial agents and chemotherapy . 52, n o 9: 3127-3134. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Table 1. S usceptibili ty of E. coli to se veral anti biotics Ant ibiotic E. coli Susceptibility Diameter/mm E 0 CX 0 RA 0 KF 0 AM 0 P 0 F 6 TE 11 SXT 20 CTX 20 TOB 15 L 10 C 23 N 26 NA 26 CN 24 0 = Resistan t, Erythromycin ( E), Cloxacellin (CX) , Rifampin (RA), Cephalo thin (K F), Ampicillin (AM), Pen icillin (P), Nitrofurantion (F), Tetracyclin e (TE), Trimethoprime + Sulfamethorazole (SXT), Cephotaxime (CTX), Tobramycin (TOB), Lincomycin (L), Chloramphenicol (C), Neomy cin (N), Nalidixic Acid (NA), Gentamicin (CN). Table 2. S usceptibili ty of E. coli to omeprazole Ome prazole μg/ml E. coli Growth 10 0 + 30 0 + + = Growth. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.23 (3 ) 2010 Table 3. Suscepti bili ty of E. coli to se ve ral an ti bi otics in ome prazole containin g me dium A n ti bi o ti c E. coli Susceptibility Diameter/mm Control Omepra zole -μg/ml 100 300 E 0 0 0 CX 0 0 0 RA 0 0 0 KF 0 0 0 AM 0 0 0 P 0 0 0 F 6 6 0 TE 11 7 0 SXT 20 15 15 CTX 20 15 10 TOB 15 15 9 L 10 11 9 C 23 15 11 N 26 18 10 NA 26 28 26 CN 24 20 9 0 = Resistant, Erythromycin ( E), Clo xacellin ( CX) , R ifampin (RA), Cephalothin ( KF), Ampicillin (AM), Penicillin (P), Nit rofurantion (F), Tetracycline ( TE), Trim ethoprime + Sulfam ethorazol e ( SXT), Cephotaxime ( CTX), Tobramycin ( TOB), Lin comycin ( L), Chloramph eni col ( C), Neomycin ( N), Nalidixic Acid ( NA), Gentamicin ( CN). Tab le 4. Molecular weight of used antib iotics (en.wikipedia.org) Antibiotic Molecular Weight g/mol RA 882.940 E 733.930 N 614.644 SXT 543.599 CN 477.596 TOB 467.515 CTX 455.470 TE 444.435 CX 435.880 L 406.538 KF 396.440 P 350.391 AM 349.406 C 323.132 F 238.160 NA 232.235 Rifampin (RA), Erythromycin (E), Neomycin ( N), Trimethoprim e + Sulfam ethorazol e (SXT), Gentamicin ( CN), Tob ram ycin ( TOB), Cephotaxime ( CTX), Tetra cycline ( TE), Cloxa cellin ( CX) , Lin com ycin ( L), Cephalothin ( KF), Penicillin ( P), Ampicillin ( AM), Chlorampheni col ( C), Nit rofurantion (F), Nalidixic Acid ( NA). IHJPAS 2010) 3( 23مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد E. coliفي الحیاتیة ومبرازول في تعزیز مقاومِة المضاداتدوراأل سراء عطیة عجیلأ، ریا رعد جبرائیل جبري، البكريعبد الرضاصالح جیھالجامعة التكنولو، قسم العلوم التطبیقیة، حیائیةفرع التقنیات األ الخالصة منتظمـة و، مسـتعمراتھا دائریـة و،عصـویة سـالبة لملـون كـرامبكتریـا :عتماد على صفاتھا التشخیصـیة باأل E.coliبكتریا عزلت ــيء و،المظھـر لقـة متأو،الحـواف ى قلیلـة اللزوجـة لزجـةو،سـمیكة بعـض الش وز و و،اـل ــرة لالكـت ود بكتریـا مخم ــة تـع ى عائل ھـذه العزلـة اـل Enterobacteriaceae. ار الحساسـیة ضـد المضـادات الحیاتیـةأظھرت نتائج اخت أن ـب ت حساسـة ل E. coliـب عكاـن المسـتعملة عـدا المضـادات الحیاتیـة جمـی نیالسیفالوث و ،)Rifampin (RA)(الرفامبین و،(Cloxacellin (CX))و كلوكساسلین ، )Erythromycin (E)(رثرومایسین ألا )Cephalothin(KF) (، ـلین Amp(و االمبس icillin (AM ـلین ، )( ــیة) . Penicillin (P)(و البنس ار حساس و قـد بینـت تجربـة أختـب E. coli ــــة ــ ــ ــ ـــز مختلـف ــ ــ ــ ــ ـــى تراكی ــ ــ ــ ــ ـــة عل ــ ــ ــ ــ ـــة حاوی ــ ــ ــ ــ ـــاط زرعی ــ ــ ــ ــ ــي أوس ــ ــ ــ ــ ـــة ـف ــ ــ ــ ــ ـــادات الحیاتی ــ ــ ــ ــ ــد المض ــ ــ ــ ضـــ omepمن دواء األومبرازول razole ،ون الل قلـة حساسـیة ،الدواء المثبط لضـخ البروـت لـى المضـادات أ E. coli تعزیـز المقاومـة مـن ـخ .التي كانت حساسة لھا عكسیا مع تركیز الدواء omepومبرازول األوضحت النتائج ان أ razole ي د غیّـَر إمكانیـةَ الغشـاِء الخلـوِي ـف ن خـالل إزالـِة إسـتقطاب غشـاِء E. coliـق ـم .الى داخل الخلیة البكتریة الحیاتیةمما عرقل نفوذیة المضادات ، ونَ الخلیِة نتیجة لتثبط عمل آلیِة ضخِّ البروت IHJPAS