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-Bakri, R. R. Jabri, E. A. Ajeel Department of Biotechnology, School of Applied Science, University of Technology. Abstract E. coli was isolated, and it was Gram-negative rod bacteria that was colony circular, regular edged, thick somewhat glitter and viscous(less). It was lactose fermenter bacteria and belongs the family of Enterobacteriaceae. E. coli showed sensitivity to all used antibiotics except Erythromycin (E), Cloxacellin (CX), Rifampin (RA), Cephalothin (KF), Ampicillin (AM ), and Penicillin (P). The experimental results of antibiotic sensitivity of E. coli in media containing different concentrations of omeprazole, a proton pump inhibitor, showed an enhancement of resistance by decreasing the sensitivity of E. coli inversely with drug concentration against the antibiotics that E. coli was sensitive to. It seems that omeprazole changed cell membrane potential of E. coli which led to depolarization of cell membrane as a result of inhibition of the proton pump mechanism. This made the bacterial cell not willing 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, nonsporing, facultative rod that ferments lactose with gas formation within 48 hour, at 35°C [2]. E. coli is the best studied bacterium and the experimental 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 (UTI). UTI is treated by antimicrobial drugs that destroy pathogenic microorganisms at low concentration called Minimum Lethal Concentration (MLC) or inhibit their growth at low concentration called Minimum Inhibitory Concentration (MIC) [5,6]. Over the past several years, the medical community has become increasingly concerned over the ability of certain bacteria to develop resistance to antibiotics [7,8]. Accordingly, there is a danger of losing the battle against certain pathogens (disease causing organisms) by using the antibiotics in the treatment[7]. Bacteria do not become resistant to antibiotics without presence of mechanisms by which resistance may be conferred [9,10]. There are four main mechanisms by which microorganisms can exhibit resistance to antimicrobials which: drug inactivation or modification, alteration of target site, alteration of metabolic pathway, or reducing drug accumulation [7]. Furthermore, there are several means through which the uptake of a drug into a cell can be reduced: changes in the structure of the cell membrane, loss, or mutations of porins in the cell membranes, and active efflux of the drug from the cell [11,12]. The drug efflux systems are membrane transport proteins. Proton motive force (pmf) system is one of the drug efflux mechanisms, is principally found in bacteria and yeasts. The pmf is also known as the electrochemical proton gradient, and a chemical proton gradient. It functions as antiporter, and therefore mediates drug efflux in exchange with proton translocation into the cell [13]. Current study dealt with the role of proton motive force mechanism in drug efflux pump in E.coli, a bacterial model, by using omeprazole, an inhibitor of pmf mechanism. Omeprazole is one of the most widely prescribed 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 dyspepsia, peptic ulcer disease (PUD), gastroesophageal reflux disease (GORD/GERD) and Zollinger-Ellison syndrome [14]. Materials and Methods Isolation of E. coli: An E. coli was isolated from patient with UTI, cutivated in Macconkey agar (HIM EDIA) plate, and then cultivated in Nutrient agar (HIM EDIA), and then its shape was identified [15]. Antibiotic Sensitivity (Disk Diffusion Method): Antibiotic sensitivity for isolated bacteria was done by using a combination of antibiotics disks (Bioanalyse); including Cloxacellin (CX) 1μg, Erythromycin (E) 15μg, Tetracycline (TE) 30μg, Ampicillin (AM ) 10μg, Gentamicin (CN) 10μg, Nalidixic Acid (NA) 30μg, Cephalothin (KF) 30μg, Neomycin (N) 30μg, Penicillin (P) 10μg, Lincomycin (L) 2μg, Cephotaxime (CTX) 30μg, Trimethoprime 1.25μg + Sulfamethorazole 23.75μg (SXT) 25 μg, Chloramphenicol (C) 30μg, Nitrofurantion (F) 300μg, Rifampin (RA) 5μg, and Tobramycin (TOB) 10μg. These disks were stored at 4°C using disk diffusion method as the following: 0.1ml of each strain was cultured by spreading on the suface of nutrient agar plate, and then antibiotic disks were placed on the surface of agar using sterile forceps and incubated 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]. Sensitivity to Omeprazole: Two different concentrations of omeprazole (stock solution of omeprazole of 1mg/ml. was prepared by dissolving the granules of 1 capsule [20mg] of omeprazole [Ajanta] in 20ml of distilled water and stored at -20°C) were prepared (100 and 300 μg/ml), and each concentration was added to 20ml of Nutrient 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 omeprazole different concentrations was detected. [17,18]. Testing the Role of Omeprazole in Antibiotic Sensitivity: Each of the prepared concentrations of omeprazole (100 and 300 μg/ml) was added to 20 ml of Nutrient agar and left to solidify. E. coli was inoculated on the surface of each plates of Nutrieint agar (the plates that contain omeprazole). The antibiotic disks (same antibiotic disks that were used in antibiotic sensitivity test) were added to the surface of Nutrient agar of each plate. Plates were incubated at 37°C for 24 hour. The results were detected and recorded according to formation, absence, increasing, or decreasing of inhibition zone around the disk .[17,18]. Results and Discussion Isolation of E. coli: E. coli was purified by cultivation on MacConkey agar media, the morphological characteristics and properties of E. coli were determined as: circular, regular edge, thick somewhat, glitter pink, viscous, and lactose fermenter [2]. Antibiotic Sensitivity: The sensitivity to antibiotics was determined and the results indicated that E. coli was sensitive to 10 antibiotics and resistant to 6 antibiotics from 16 types of antibiotics used (Table 1). These results were used as a control for further comparison (Table 3). Sensitivity to Omeprazole: The recommended dosages for patients taking omeprazole are 10mg, 20mg, or 40mg. But the most frequent side effects of omeprazole are headache, diarrhea, abdominal pain, nausea, dizziness, 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 cardiac arrest [21]. The study dealt with minimum concentrations (100, and 300 μg/ml) (MIC) of omeprazole to minimize its side effect on human and ensure its activity on E. coli. (17,18). E. coli was cultivated in each concentration of omeprazole containing agars after 24 hr. of incubation at 37°C. The study found that E. coli grew at both omeprazole concentrations (Table 2) and this drug showed no killing effect on E. coli [22,23]. For this reason this IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.23 (3) 2010 research used omeprazole to find out its role in modulation of antibiotic resistance in E. coli. Since omeprazole is a proton pump inhibitor [13,22]. Omeprazole - Antibiotic Sensitivity 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 were obvious decreasing in the diameters of inhibition zones of the most antibiotic types that E. coli was sensitive to reversely with omeprazole concentrations as compared with control group in (Table 3). In addition, E. coli exhibited its resistance against TE and F at 300μg/ml of omeprazole. Accordingly, the total antibiotic types that E. coli was resistant to, had increased at 300μg/ml of omeprazole containing medium to become 8 from 16 antibiotic types (Table 3). These results proposed that omeprazole had an important role in the enhancement of resistance of E. coli against antibiotics [24]. Omeprazole is a proton pump inhibitor [13,22]. The proton pump system is an integral membrane protein that is capable of grabbing protons from the matrix (the space enclosed by the two membranes) and releasing the protons into the inter-membrane space. The confined protons create a difference or gradient in both pH and electric charge and establish an electrochemical potential. Because there's a higher concentration of protons in the inter-membrane space compared to inside the cell, there's pressure to return protons down the concentration gradient to restore the balance. This pressure is called the proton motive force (pmf) [25]. In current study, after treatment of E. coli with omeprazole, a gradient in electric charge might be created, the concentration of protons inside the bacterial cell was higher than its concentration in the inter-membrane space [26]. There was no way to restore the balance by pumping the protons outside the cell [27] due to the action of omeprazole as a proton pump inhibitor. Blocking the passage of proton pumping seemed to create a decreasing in cellular membrane potential (Depolarization of cell membrane) [28]. Depolarization of cell membrane may make E. coli not willing to uptake the antibiotics into the cell [29]The results of antibiotic sensitivity in omeprazole containing media give an indirect suggestion that there were no effect of gradient in pH [30] on the downsizing of porins to make the permeability of cell membrane selectable [31] for the antibiotics of low molecular weight [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 resistance or decreasing the sensitivity against them after the treatment with 100 μg/ml omeprazole 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 omeprazole containing media. These results are in agreement with the study of Perlin, and his colleagues [24]. They reported the electrogenic behavior of proton transport by the H + -ATPase in Saccharomyces cerevisiae. H +-ATPase is encoded by pma1 gene. The study found that mutations within pma1 may alter steady-state membrane potential formation, possibly through a change in the electrogenicity of the H + -ATPase. This make the mutant exhibited its resistance to hygromycin B that may be mediated via depolarization of the cellular membrane potential. Conclusion The experimental results revealed that the treatment of E. coli with omeprazole, a proton pump 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 that E. coli was sensitive to (before the treatment with omeprazole). The study suggested that the decreasing in the sensitivity may mediate by depolarization of cellular membrane via a potential change in cell membrane as a result of proton pump inhibition by omeprazole. Accordingly, the modulation of antibiotic resistance by a proton pump inhibitor is not recommended because the treatment of bacterial infection may become more complicated. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (3) 2010 References 1. Ewing, W.H. (1986). “Edwards and Ewing's Identification of Enterobacteriaceae”, 4th ed. Elsevier, New York. 2. Feng, P., Weagant, S.and Grant, M. 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National Committee for Clinical Laboratory Standards. (1997). Specialty Collection: Susceptibility Testing. SC21-L. M7-A4. NCCLS, Wayne, PA. 19. rxlist.com, (2007). "Prilosec Side Effects & Drug Interactions".WWW.healthcentral-Cem GERD/ /Heartbum 20. Yang, Y. X., Lewis, J.D., Epstein, S., Metz, D. C. (2006). "Long-term Proton Pump Inhibitor Therapy and Risk of Hip Fracture". JAMA 296 24: 2947–53. 21. Cunningham ,R., et al. (2003). “Proton pump inhibitors as a risk factor for Clostridium difficile diarrhea”. Journal of Hospital Infection 54: 243-245. Bandolier:http://www.medicine.ox.ac.uk/bandolier/booth/Pharmacy/PPIcdiff.html 22. Kuhler, T., Fryklund, J., Bergman, N. A., Weilitz, J., Lee, A., and Larsson, H. (1995). “Structure-activity relationship of omeprazole and analogues as Helicobacter pylori urease inhibitors”. J. Med. Chem. 38: 4906-4916. 23. Bayerdoerffer, E., Mannes, G. A., Sommer, A., Hoechter, W., Weingart, J., Hats, R., Lehn, N., Ruckdeschel, G., Dirschedl, P., and Stolte, M. (1992). “High dose omeprazole treatment combined with amoxicillin eradicates Helicobacter pylori”. Eur. J. Gastroenterol. Hepatol. 4:697-702. 24. Perlin, David S., Brown, Colette L., and Haber, James E. (1988). “Membrane Potential Defect in Hygromycin B-resistnat pma1 Mutants of Saccharomyces cerevisiae”. The Journal of Biological Chemistry. 263, 34: 18118-18122. 25. Nicholls D, Ferguson S. (1992).”Bioenergetics 2”. London,Academic Press. 26. Nagle, J.F., and H.J. Morowitz. (1978). “Molecular mechanisms for proton transport in membranes”. Proc. Natl. Acad. Sci. USA. 75:298-302. 27. DeCoursey, T.E. (2003). “Voltage-gated proton channels and other proton transfer pathways”. Physiol. Rev. 83:475-579. 28. Thomas, R.C., and R.W. Meech. (1982). “Hydrogen ion currents and intracellular pH in depolarized voltage- clamped snail neurons”. Nature. 299:826-828. 29. Erdogan, Seref; Ozgunen, Kerem Tuncay; Ozgunen, Tuncay. (2004). “Effects of H+-K+ ATPase Inhibitors (Omeprazole and Lansoprazole) on Fertilization-Induced Bioelectrical Potential Changes in the Egg of the Frog, Rana cameranoi”. Turk J. Vet. Anim. Sci. 28: 329-336. 30. Demaurex, N., Grinstein, S., Jaconi M., Schlegel, Lew,W. D.P. and K. H. Krause. (1993). “Proton currents in human granulocytes: regulation by membrane potential and intracellular pH”. J. Physiol. (Lond.). 466:329-344. 31. Todt, Jell C., Rocque, Warren J., and McGroarty, Estelle J. (1992). “Effects of pH on Bacterial Porin Function”. Biochemistry. 31: 10471-10478. 32. Danilchanka, Olga; Pavlenok, Mikhail;and Niederweis, Michael (2008). “Role of Porins for Uptake of Antibiotics by Mycobacterium stnepnatik”. Antimicrobial agents and chemotherapy. 52, n o 9: 3127-3134. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (3) 2010 Table 1. Susceptibility of E. coli to several antibiotics Antibiotic 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 = Resistant, Erythromycin (E), Cloxacellin (CX) , Rifampin (RA), Cephalothin (KF), Ampicillin (AM), Penicillin (P), Nitrofurantion (F), Tetracycline (TE), Trimethoprime + Sulfamethorazole (SXT), Cephotaxime (CTX), Tobramycin (TOB), Lincomycin (L), Chloramphenicol (C), Neomycin (N), Nalidixic Acid (NA), Gentamicin (CN). Table 2. Susceptibility of E. coli to omeprazole Omeprazole μg/ml E. coli Growth 100 + 300 + + = Growth. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.23 (3) 2010 Table 3. Susceptibility of E. coli to several antibiotics in omeprazole containing medium An tibi otic E. coli Susceptibility Diameter/mm Control Omeprazole -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), Cloxacellin (CX) , R ifampin (RA), Cephalothin (KF), Ampicillin (AM), Penicillin (P), Nit rofurantion (F), Tetracycline (TE), Trimethoprime + Sulfamethorazole (SXT), Cephotaxime (CTX), Tobramycin (TOB), Lincomycin (L), Chloramphenicol (C), Neomycin (N), Nalidixic Acid (NA), Gentamicin (CN). Table 4. Molecular weight of used antibiotics (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), Trimethoprime + Sulfamethorazole (SXT), Gentamicin (CN), Tobramycin (TOB), Cephotaxime (CTX), Tetracycline (TE), Cloxacellin (CX) , Lincomycin (L), Cephalothin (KF), Penicillin (P), Ampicillin (AM), Chloramphenicol (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 ى قلیلـة اللزوجـة لزجـةو،سـمیكة بعـض الشــيء و،المظھـر لقـة متأو،الحـواف وز و و،اـل ود بكتریـا مخمــرة لالكـت 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 Ampicillin (AM(و االمبس ـلین ، )( ار حساســیة) . Penicillin (P)(و البنس 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 ون، omeprazoleمن دواء األومبرازول الل قلـة حساسـیة ،الدواء المثبط لضـخ البروـت لـى المضـادات أ E. coli تعزیـز المقاومـة مـن ـخ .التي كانت حساسة لھا عكسیا مع تركیز الدواء ي omeprazoleومبرازول األوضحت النتائج ان أ 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