Efficacy of combination Meropenem with Gentamicin, and Amikacin against resistant E Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 66 Efficacy of Combination of Meropenem with Gentamicin, and Amikacin against Resistant E. coli Isolated from Patients with UTIs: in vitro Study # Maysaa A. abdul khaleq* ,1 , Abdulkareem H. Abd** and Maysaa A. dhahi*** * Department of pharmacotheraputics,College of Pharmacy, Al- Mustansiryh University,Baghdad,Iraq. ** Department of pharmacology, College of Medicin, Al- Nahrain University, Baghdad, Iraq. *** Department of Microbiology, College of Medicin, Al-Nahrain University, Baghdad, Iraq. Abstract Seventy five E. coli isolates were collected from urine of patients with urinary tract infections in AL-Kadhimia and AL-Yarmook teaching hospitals in Baghdad for a period between 22/11/2009 to 15/3/2010, from these samples twenty five isolates were selected according to their pattern of the highest resistance as these showing multi-drug resistances and tested to specify their minimum inhibitory concentration for (meropenem, gentamicin and amikacin), meropenem was found having the lowest MIC comparing with others. This study also includes in vitro effects of various combinations of three types of antimicrobials (meropenem, gentamicin and amikacin) against twenty five E. coli isolates.Among combinations the combination of meropenem with the other types of antimicrobials showed high synergistic effect when 1/4+1/4 MIC for each antimicrobial were used. While combinations of amikacin with gentamicin in some isolates showed additive effect when 1/2+1/2 MIC for each antimicrobial were used. The plasmid profile for the twenty five E. coli isolates were studied using Pure Yeild ™ plasmid Miniprep system- Cat.# A1220 – Promega- USA. In order to determined the presence of plasmid for antimicrobials resistance. الخالصة جَعج خَست ٗسبعُ٘ عضىت ٍِ االشٞشٝشٞا اىق٘ىّ٘ٞت ٍِ ادساس ٍشضٚ اىَجاسٛ اىب٘ىٞت اىزِٝ ساجع٘ا ٍسخشفٚ اىناظَٞت عيٚ ٍا ٌٍْٖٗ حٌ اخخٞاس خَست ٗعششُٗ عضىت اعخَادا ۲۰۱۰⁄ ۳ ⁄۱۱اىٚ ۲۲⁄۱۱⁄۲۰۰۰ٗاىٞشٍ٘ك اىخعيَٜٞ فٜ بغذاد ىيفخشٓ ٍِ اىجْخَاٝسِٞ ،ىيَضاداث ) اىَٞشٗبٌْٞ (MIC)اداث اىجشثٍ٘ٞت ثٌ حذدث اىخشامٞض اىَثبطت اىذّٞا بذحٔ ٍِ ٍقاٍٗت عاىٞت ٗ ٍخعذدة ىيَضٴ ا قو حشمٞض ٍقاسّت باىَضاداث ٴ مثش فاعيٞت ٗ رىل بخثبٞطٔ َّ٘ اىبنخشٝا باٴ ظٖشث اىْخائج باُ ٍضاد اىَٞشٗبٌْٞ ٕ٘ االٴ ٗقذ ا )ٗاالٍٞناسِٞ ٗقذ E. coli ((in vitroححاد اىَضاداث اىحٞ٘ٝت ضذ خَست ٗعششُٗ عضىت ٍِ ثٞش اٴ حضَْج ٕزٓ اىذساست اسخقصاء حا.خشٙٴ اال ٝشٞش اىٚ حاثٞش حاصسٛ عاىٜ عْذ اسخعَاه )ُ اححاد اىَٞشٗبٌْٞ ٍع بقٞت اىَضاداث اىحٞ٘ٝت)اىجْخَاٝسِٞ ٗاالٍٞناسِٞ ٴ ظٖشث اىْخائج اٴ ا الٍٞناسِٞ ٍع اىجْخاٍاٝسِٞ فٜ بعض اىعضالث ٝشٞش اىٚ حاثٞش بَْٞا اححاد ا .ىنو ٍضاد حٞ٘ٛ MIC)سبع اىخشمٞض اىَثبط االدّٚ ) ىَْط اىبالصٍٞذٛ ٴ دساست ا ىذساست اٝضا ٴ شَيج ا .ىنو ٍضاد حٞ٘ٛ MIC)اضافٜ فقط عْذ اسخعَاه ّصف اىخشمٞض اىَثبط االدّٚ ) ُ اىعضالث ٴ ظٖشث اىْخائج باٴ ٗقذ ا Miniprepسخخذاً عذة ىعضه اىبالصٍٞذ ب٘اسطت ّظاً ٴ با E. coliىخَست ٗعششُٗ عضىت ٍِ بنخشٝا .ىَضاداث اىحٞ٘ٝتٴ ( حاٍئ ىبالصٍٞذ ىَقاٍٗت ا٬،۳۲،۳۳،۱۳) Introduction Urinary tract infections (UTIs) are one of the most common bacterial infections in humans both in the community and hospital setting (1) . Escherichia coli have been documented to be the most important pathogen associated with symptomatic urinary tract infections (2) .plasmid DNA molecule is separate from, and can replicate independently of, the chromosomal DNA (3) . In this study we use combination of meropenem (which is a broad spectrum antimicrobial agent with more activity against gram-negative bacilli and less activity against gram-positive cocci than is imipenem) (4) , with aminoglycosides which are polar compound with more activity against aerobic gram-negative bacilli and little activity against an aerobic bacteria and use with other antimicrobial agent against gram positive bacteria (5) . # Based on oral presentation in the eighth scientific conference of the College of Pharmacy /University of Baghdad held in 23-24 February 2011. 1 Corresponding author E- mail : maysaa_ali82a@yahoo.com Received : 12/3/2011 Accepted : 10/5/2011 Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 67 Material and Methods The E. coli identification depended on morphological, biochemical testes in addition to API 20E system. Susceptibility of isolates to seventeenth antimicrobials was tested using disk diffusion assay according to modified Kirby–Bauer method (6) . Meropenem, nitrfurantoin , amikacin and imepenem were to be the most effective antimicrobials, while the other antimicrobials were less effective. Minimum inhibitory concentration (MIC) was determined using tubes dilution method (7) . The combination of antimicrobials weather it’s synergistic, additives, antagonistic, or indifference depending on the fractional inhibitory concentration (FIC) was determine as follow: (≤0.5) synergism, (0.5–<1) additive, (1–<4) indifference,(≥4) antagonism, and calculated using the following equation (8) . MIC for antibiotic in combination FIC = ———————————————–– MIC for antibiotic alone Plasmid DNA isolated using Pure Yeild ™ plasmid Miniprep system, according to the manufacture manual. Then the extracted plasmid DNA was loaded in 0.8% agarose gel stained with ethidium bromide and electrophoresis for 60 minutes at 2V/Cm using 1X TBE buffer. Then agarose gel was visualized using UV-transluminator. Result and Discussion Colonies of E. coli had marked as a flat smooth and pink in color as a result of lactose fermentation in the media on MacConky agar, while on blood agar it gave small pink convex colonies surrounded by zone of β- haemolysis. In Microscopic Examination it showed as small single bacilli non spore forming with red color (gram –negative bacteria), it occurred separately and singly, but often they are accumulated in groups. The result of biochemical tests for most of E. coli showed its ability to catalase production and lactose fermentation while it gave a negative result in Oxidase, Urease and Simmon Citrate tests. Further identification of the isolates was done by using Api 20E system, as in Figure (1). E. coli (4) Figure 1: Identification of E. coli by Api20E system Antimicrobial Sensitivity Test 1-Qualitative Method (Disc Diffusion Test) In this study we found that antimicrobials sensitivity among E. coli isolates varied according to the nature of antimicrobials. The percentage of resistant isolates to each antimicrobial is shown in Figure (2).Standard disc diffusion assay was used to detect the sensitivity of pathogenic bacteria and results obtained were compared with those of Clinical and laboratory standard institute (9) .The results of the current study (Figure 2) revealed that most of E. coli isolates resist the β- lactam antimicrobials (like ampicillin and amoxicillin) (10) .noted the high resistance rates of gram positive and gram negative species to penicillins and some of cephalosporins. Increasing of bacterial resistance rates to this group of antimicrobials may be a result of either production of β- lactamase enzyme that had the ability to destroy the β- lactam ring in these antimicrobials (11, 12) . Also it may be due to minimizing the interaction of antimicrobials with target site (Penicillin Binding Proteins) (13) .Augamentin ( amoxicillin + clavulanic acid) had more activity than other penicillin due to its presence of clavulanic acid, which inhibit β- lactamase enzyme, and increase the spectrum of amoxicillin against gram- positive Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 68 and gram- negative bacteria (14) . Many research illustrated the higher activity of imipenem and meropenem (related to carbapenems group) against gram- positive and gram- negative bacteria (15) .Regarding aminoglycoside group, amikacin was more active than gentamicin on the current E. coli isolates, many researches showed that the increasing resistance against aminoglycoside group was due to production of the modified enzymes and losing outer membrane pores, which are responsible of permeability of surface cell layer to antimicrobials (16) . The current results (Figure 2) was in agreement with that of Shevelev et al. (2002) (17) who found in a study that the resistance percentage of the isolates to amikacin was (0%) , while the resistant rate to gentamicin was (48.6%). The results also was in agreement with Bashir et al.( 2008) (18) who found in a study in Pakistan that the resistance percentage of the isolates to gentamicin was (49%) . Resistant to tobramycin was (40.7%) and this result was near that found by Pape et al. (2004) (19) who found that the resistant percentage of E. coli to tobramycin was (30%).Many studies were illustrated the activity of naldixic acid, and most of quinolones antimicrobials against wide range of bacteria that were in a good agreement with the currently result. For example the resistant rate to ciprofloxacin was (40.7%) this result was comparable to the result of Shamm et al.(2001) (20) found in a study that the resistant percentage of E. coli to ciprofloxacin was (39%). Resistance to pipracillin was (85.5%), this result was in agreement with that of Bujdakova et al.(1998) (21) who found that (86%) of E. coli isolates resistant to pipracillin , and this may be due to the ability of E. coli to develop resistance to these antimicrobials through the production of β-lactamase enzyme which break the β-lactam ring of pipracillin. Resistance to nitrofurantoin was (2.6%), this result was in agreement with Akyar (2008) (22) who found that the resistant rate of E. coli against nitrofurantoin was (3%).Resistance to trimethoprim/ sulfamethoxazole (SXT) was (43.4%), this result may be attributed to the wide use of (SXT) as empirical therapy for urinary tract infection, however this result was in agreement with Gupta; Hooton and Stamm (2001) (23) who found that the resistance to (SXT) among E. coli isolates from patient with UTIs has increased, with a prevalence of resistance which is reported 30 to 50 percent . Figure 2: Percentage of resistant E. coli isolates to antimicrobial ToB: Tobramycin; CN: Gentamicin; Sxt: Triomethoprime and sulfamethoxazole; Cip: Ciprofloxacin; Na: Naldixic acid; Ctx: Cefotaxime; Ipm: Imipenim;Am: Ampicillin; CL: Cephalexin; CRO:Ceftriaxone;AMC:Amoxicillin and Clavulonicacid; F:Nitrofurantoin; AZM:Azithromycin;PRL:Pipracillin;MPM: Meropenem; AX:Amoxicillin AK:Amikacin 2- Quantitative Method (Minimum Inhibitory Concentration) (MIC) Table 1 showed that MIC of meropenem ranged from (0.003-12.5μg/ml) this result was in agreement with Marie et al. (24) who found in his study that E. coli was moderately susceptible to meropenem at MIC (8μg/ml) .The results of this study also showed that the MIC of gentamicin ranged from (12.5 to 480 μg/ml), this result was in agreement with Jakobsem et al. (25) who found in his study that the MIC of gentamicin distributed from (8-› 512 μg/ml)..On the other hand MIC of Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 69 amikacin ranged from (0.3-2.5μg/ml), this result was in agreement with Shrivastava and Chaudhary (26) whose found that the MIC of amikacin in E. coli was (2μg/ml).while Celine et al. (27) who found in his study that the MIC of amikacin in E. coli ranged from (1 to16 μg/ml). Table 1: MIC value for three antimicrobials (µg/ml) tested against E. coli isolates E. coli isolates Meropenem µg/ml Gentamicin µg/ml Amikacin µg/ml MIC MBC MIC MBC MIC MBC A1 0.12 0.125 300 300 2.5 5 A2 1.25 12.5 200 300 1.25 2.5 A3 0.12 1.25 300 300 1.25 2.5 A4 1.25 1.25 300 480 0.6 1.25 A6 0.12 1.25 480 480 0.3 0.6 A7 1.25 1.25 300 300 2.5 5 A10 12.5 12.5 480 480 0.6 1.25 A11 0.003 0.003 12.5 12.5 1.25 2.5 A13 0.12 0.12 300 300 2.5 5 A24 0.12 1.2 300 300 1.25 2.5 A28 0.03 0.03 200 200 1.25 2.5 A32 12.5 12.5 480 480 0.6 1.25 A35 1.25 1.25 200 300 0.6 1.25 A37 0.12 1.25 480 480 0.3 0.6 A41 12.5 12.5 100 200 1.25 2.5 A42 12.5 12.5 100 200 1.25 2.5 A43 1.25 1.25 200 300 0.6 1.25 A44 0.06 0.06 200 300 2.5 5 A45 1.25 12.5 200 300 1.25 2.5 A47 0.12 1.25 480 480 0.3 0.6 A51 12.5 12.5 100 200 1.25 2.5 A55 0.06 0.03 200 300 2.5 5 A57 0.12 1.25 480 480 0.3 0.6 A58 0.12 0.12 300 300 2.5 5 A67 0.12 0.125 300 300 2.5 5 LSD value 4.945 * 5.418 * 137.95 * 118.38 * 0.830 * 1.651 * * (P<0.05), LSD: Least significant difference 3- Antimicrobials Combination The result in Table2 shows that the synergistic effect noticed from combination of meropenem with gentamicin on isolate No. (1, 2, 3, 4, 6, 7, 10, 13, 24, 28, 35, 37, 41, 42, 43, 44, 45, 47, 51, 55, 57, 58, 67), this result similar to that shown by Richared et al. (28) found that aminoglycoside synergized with β- lactams antimicrobials against E. coli isolates, because of the latter action on cell wall synthesis, which enhance diffusion of the aminoglycoside into the bacterium. While isolate No.(32) show the additive effect with combination of meropenem with gentamicin, and that may be due to their resistance to gentamicin (MIC 480) and to meropenem (MIC 12.5). table3 shows Another synergistic effect resulted from combination of meropenem with amikacin when its effect tested on isolate No. (1, 2, 3, 4, 7, 10, 13, 24, 28, 37, 41,42,43, 44,45,47, 51, 55, 57, 58, 67) this result was in agreement with and Piroska et al. (29) whose found that there is synergistic effect result from combination of meropenem with amikacin against E. coli isolates . While isolates No. (6, 32, 35) showed no effect toward combination of meropenem with amikacin.On the other hand combination of amikacin with gentamicin ( table 4 showed additive effect when tested on isolates No. (1, 2) but other isolates show no effect. Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 70 Table2: Results of combination of meropenem with gentamicin (1/4+1/4MIC) Result FIC MIC of gentamicin after combination (ug/ml) MIC of gentamicin before combination (ug/ml) MIC of meropenem after combination (ug/ml) MIC of meropenem before combination (ug/ml) E. Coli isolates Syn 0.5 75 300 0.03 0.12 A1 Syn 0.5 50 200 0.31 1.25 A2 Syn 0.5 75 300 0.03 0.12 A3 Syn 0.5 75 300 0.31 1.25 A4 Syn 0.5 120 480 0.03 0.12 A6 Syn 0.5 75 300 0.31 1.25 A7 Syn 0.5 120 480 3.125 12.5 A10 Syn 0.5 75 300 0.03 0.12 A13 Syn 0.5 50 200 0.007 0.03 A28 Syn 0.5 75 300 0.03 0.12 A24 Syn 0.5 50 200 0.31 1.25 A35 Syn 0.5 120 480 0.03 0.12 A37 Syn 0.5 25 100 3.12 12.5 A41 Syn 0.5 25 100 3.12 12.5 A42 Syn 0.5 50 200 0.31 1.25 A43 Syn 0.5 50 200 0.015 0.06 A44 Syn 0.5 50 200 0.31 1.25 A45 Syn 0.5 120 480 0.03 0.12 A47 Syn 0.5 25 100 3.12 12.5 A51 Syn 0.5 50 200 0.01 0.06 A55 Syn 0.5 120 480 0.03 0.12 A57 Syn 0.5 75 300 0.03 0.12 A58 Syn 0.5 75 300 0.03 0.12 A67 -- -- 122.23 * 213.56 * 4.234 * 5.030 * LSD value * (P<0.05); LSD: Least significant difference; Syn: Synergism; FIC: Fractional Inhibitory Concentration Table 3: Results of combination of meropenem with amikacin (1/4+1/4 MIC): Result FIC MIC of amikacin after combination (ug/ml) MIC of amikacin before combination (ug/ml) MIC of meropenem after combination (ug/ml) MIC of meropenem before combination (ug/ml) E. Coli isolates Syn 0.5 0.62 2.5 0.03 0.12 A1 Syn 0.5 0.31 1.25 0.31 1.25 A2 Syn 0.5 0.31 1.25 0.03 0.12 A3 Syn 0.5 0.15 0.6 0.31 1.25 A4 Syn 0.5 0.62 2.5 0.31 1.25 A7 Syn 0.5 0.15 0.6 3.12 12.5 A10 Syn 0.5 0.62 2.5 0.03 0.12 A13 Syn 0.5 0.31 1.25 0.03 0.12 A24 Syn 0.5 0.31 1.25 0.007 0.03 A28 Syn 0.5 0.07 0.3 0.03 0.12 A37 Syn 0.5 0.31 1.25 3.12 12.5 A41 Syn 0.5 0.31 1.25 3.12 12.5 A42 Syn 0.5 0.15 0.6 0.31 1.25 A43 Syn 0.5 0.62 2.5 0.01 0.06 A44 Syn 0.5 0.31 1.25 0.31 1.25 A45 Syn 0.5 0.07 0.3 0.03 0.12 A47 Syn 0.5 0.31 1.25 3.12 12.5 A51 Syn 0.5 0.62 2.5 0.01 0.06 A55 Syn 0.5 0.07 0.3 0.03 0.12 A57 Syn 0.5 0.62 2.5 0.03 0.12 A58 Syn 0.5 0.62 2.5 0.03 0.12 A67 -- -- 122.23 * 213.56 * 4.234 * 5.030 * LSD value * (P<0.05); LSD: Least significant difference; Syn: Synergism; FIC: Fractional Inhibitory Concentrations Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 71 Table 4: Antimicrobials combination (1/2+1/2 MIC for each antimicrobials) E. coli isolates Antimicrobials combination MIC of first antimicroal alone (µg/ml) MIC of first antimicrobial in combination (µg/ml) MIC of second antimicrobial alone (µg/ml) MIC of second antimicrobial in combination (µg/ml) FIC Results A32 MPM+CN 12. 5 6. 25 480 240 1 Add A1 AK+CN 2. 5 1. 25 300 150 1 Add A2 AK+CN 1.25 0. 625 200 100 1 Add Add: Addition; FIC: Fractional Inhibitory Concentration MPM: meropenem; CN: gentamicin; AK: amikacin. Extraction of Plasmid DNA The result of Figures (3and 4) indicate that each of the isolates (A6 , A37)containing two bands of plasmid DNA with approximate molecular weight (2000 and 1900) bp comparing with molecular weight marker. Also, isolates No.(A32, A57) containing one plasmid DNA with approximate molecular weight (2000) bp when comparing with molecular weight marker.There are many studies referred to the isolation of antimicrobial resistance plasmid from E. coli. Joseph et al. (2001) (30) found in their study that E. coli isolates contain plasmid coding for resistance of aminoglycoside antimicrobials, including gentamicin and tobramycin. Also, March Galimand et al(2003) (31) found in their study that E. coli isolated from patient suffering from urinary tract infection contain plasmid coding high level of resistance to aminoglycoside. Piddock (1999) (32) found in his study that E. coli contain plasmid coding for resistance of flouroquinolone .Sisson et al. (2002) (33) found in their study that resistance to nitrofurantoin may be chromosomal or plasmid mediated. Minch chau phuc Nguyen et al. (34) found in their study that the plasmid gene that confers resistance to azithromycin had recently emerged in non multidrug resistant E. coli; Philippon; Arlet and Jacoby (2002) (35) found in their study that E. coli contains plasmid coding for resistance of ampicillin. In the other hand, other E. coli isolates that show no plasmid may be due to carrying plasmids with low copy number. Figure 3: plasmid profile of E. coli strains Lane (A6, A37, A57, A32): Plasmid DNA extracted from E. coli strains; M.W: Molecular weight marker of lambda DNA digested with EcoRI+HindIII . Electrophoresis was carried in 0.8% agarose gel at (2V/Cm) for 30 min. 5148-4973bp 4268bp 3530bp 2027bp 1904bp 1584bp 1375bp 947bp 831bp 564bp Chromosome Plasmids Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 72 Figure 4: plasmid profile of E. coli strains isolated from UTIs patients Lane (A6, A37, A57, A32): Plasmid DNA extracted from E. coli strains; M.W: Molecular weight marker of lambda DNA digested with EcoRI+HindIII . Electrophoresis was carried in 0.8% agarose gel at (2V/Cm) for 60 min. Reference 1. David, S.; and Howes ,MD.Urinary tract infection in female;2009;27. 2. Musa-Aisien, AS.; Ibadin ,OM.; and Ukoh ,G. Prevalence and antimicrobial sensitivity pattern in urinary tract infection in febrile under-55 at a childrenś emergency unit in Nigeria.Annual of tropical pediatric; 2003;23:39-45. 3. Lipps G. Plasmids: Current Research and Future Trends; Caister Academic Press;2008. 4. Henry, F.; and Chambers, MD. Basic and clinical pharmacology .10 th ed. In: Bertram G. and Katzung M.D. Lang medical book /Mc Graw Hill;2007; PP. 726 -770. 5. Falagas, ME.; Grammatikos, AP.; and Michalopoulos, A. Potential of old- generation antibiotics to address current need for new antibiotics. Expert. Rev. Anti. Infect. Ther;2008; 6(5): 593-600. 6. Vandepitte, J. ;and Verhaegen, J.; Engbaek, K.; Rohner, P.; Piot ,P.; Heuck ,C.C. Basic laboratory procedures in clinical bacteriology. 2 nd ed. World health organization;2003; PP.30 :109-115. 7. Michael, J.; Leboffe and Bruton, E.; Pierce. A photographic atlas for the 3ed ed. Microbiology laboratory /Douglas, N. Morton, Inc.2005. 8. American society for microbiology. Instructions to Authors.S.I.: Antimicrob. Agents chemother;2002; 46:i-xix. 9. Clinical and laboratory standard institute (CLSI) .Performance standard for antimicrobial susceptibility testing. 17 th informational supplement; Document M100-S17; Clinical and laboratory standards institute;2007;Vol.27.No.1. 10. Akther, J.; Qutub, M.; and Qadri, S. Antimicrobial susceptibility testing and patterns of resistance at tertiary care center in Saudi . Med. J.; 2001;22(7):569- 576. 11. Gupta, K.; Scholes, D.; and Stamm, W. Increasing prevalenceof antimicrobial resistance among pathogens causing acute uncomplicated cystitis in women. JAMJ;1999; 281:736-738. 12. Murray, P.R.; Baron, E.J.; Pefaller, M.A.; Tenover, F.C.; and Yolken, R.H. Manual of clinical microbiology. 7 th ed. ASM press. USA.;1999. 13. Clark,W.G.; Brater, D.C.; and Johnson, A.R. Medic pharmacology Gothś in to chemotherapy mechanisms of antimicrobial. International press;1992. 5148-4973bp 4268bp 3530bp 2027bp 1904bp 1584bp 1375bp 947bp 831bp 564bp Chromosome Plasmids Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 73 14. Normann,P.; Ronse, E.; Nass ,T.; Duport, C.; and Labia, R. Characterization of a novel extended-spectrum β- lactamase from Pseudomonas areuginosa. Antimicrob.Chemother;1993; 37(5): 962- 969. 15. Gales, AC.; Sader, HS.; And Jones, RN. Urinary tract infection trends in latin American hospitals: report from the sentry antimicrobial surveillance program (1997- 2000). Diagnostic microbiology and infect Dis.;2002;44:289-299. 16. Vatopoulos, A.; Tsakris, A.; Tzouvelekis, L.; Legakis, N.; Pitt,T.; and Komninou, Z. Diversity of aminoglycosides resistance in Enterobacter Cloacaein Greece .J. Clin.Microbiol. Infect.Dis.;1992 ; 11(2):131-138. 17. Shevelev, A.; Reshedko, G.; Edelstein, I.; Kozlova, L.; Korovina, N.;Zorkin, S.; Katosova, L.; Papajan, A.; Marusina, N.; Alumetova, L.;Vjalkova, A.; Agapova, E.;and Fokina, B.Mechanisms of resistance to aminoglycosides (amg) E. coli isolates from children with community –aquired urinary tract infections, 4 th European congress of chemotherapy and infection. Paris; France; 2002. 18. Bashir, MF.; Qazi, JI.; Ahmad, N.; and Riaz, S. Diversity of urinary tract pathogens and drug resistant isolates of Esherichia coli in different age and gender groups of Pakistanis. Tropical journal of pharmaceutical research; 2008;7(3): 1025- 1031. 19. Pape, L; Gunzer, F; Ziesing, S; Pape, A; Offiner, G; and Ehrich, JH.Bacterial pathogens, resistance patterns and treatment options in community acquired pediatric urinary tract infection. Klin padiatr; 2004;216(2): 83-86. 20. Sahm, D.F.,C.; Thornsberry, C.; Mayfield, D.C.; Jones, M.E.; and Karlowsky,J.A. Multi- drug resistant urinary tract isolates of Esherichia coli : Prevalence and patient demographics in united states in 2000.Antimicrob.Agents; Chemother;2001;45:1402-1406. 21. Bujdakova, H.; Lausova, A.; Jankovicova, S.; Prodinger, W.M.; Kallova, J.;Milosovic, P.; and Kettner, M. Study of β-lactam resistance in ceftazidime resistant clinical isolates of enterobacteriaceae. J.Antimicrob. Agents ;1998;10: 136-414. 22. Akyar, I. Antibiotic resistance rates of extended spectrum beta lactamas producing Escherichia coli and Klebsiella spp. Strains isolated from urinary tract infection in a private hospital. Microbiyol Bul. National center of biotechnology information,U.S. National library of medicine; 2008. 23. Gupta, K.; Hooton, TM.; and Stamm, WE. Increasing antimicrobial resistance and the management of un complicated community acquired urinary tract infections. Ann.Internal medicine;2001;135: 41-50. 24. Mari- Frederique; Laurent poirel; Claire poyart; Helene reglier- poupet; and Patrice nordmann. Ertapenem resistance of Escherichia coli.;2007;Vol.13.Number 2. 25. Jakobsen, L.; Sandvang, D.; Jensen, V.F.; Seyfarth, A.M.; Frimodt- Moller, N.; and Hammerum, A.M. Gentamicin susceptibility in Escherichia coli related to the genetic back ground :problems with break points. Clinical microbiology and infection. Blak well publishing;2007; Vol. 13. Number 8; PP. 830-832. 26. Shrivastava, S.M.; and Chaudhary, M.Comparative studies on susceptibility and minimum inhibitory concentration of potentox, a fixed dose combination of cefepime and amikacin in Proteus vulgaris , Escherichia coli and Bacillus subtilis. Journal of medical science;2009; Vol. 9; PP. 245-248. 27. Celine Vidaillac; Steve, N. Leonard; Helie, S. Sader; Ronald, N. Jones; and Michael, J. Rybak. "In vitro activity of ceftaroline alone and in combination against clinical isolates of resistance gram-negative pathogens, including β- lactamase- producing Enterobacteriaceae and Pseudomonas aeruginosa". Antimicrobial agents and chemo.therapy;2009; Vol. 53.No. 6PP. 2360-2366. 28. Richard ,A. Harvey; Pamela, C. Champe. Protein synthesis inhibitors; In: Richard, D. Howland ; Mary, J. Mycek.Lippincotts illustrated reviews. 3ed ed. Lippincott Williams and Wikins; 2006; PP. 363-393. 29. Piroska Anderlike; Ferenc Rozgonyia; and Kajroly Nagya. The effect of amikacin and imipenem alone and in combination against an extended– spectrum β-lactamase producing Klebsiella pneumonia and E. coli strain; 2007; Vol. 58. Issue1. PP. 105-110. 30. Joseph, W. Chow; Vivek, kAk.; IIyou;Susan, J. Kao; Joanne Petrin; Don,B. Clewell ; Stephen, A. Lerner; George, H. Miller; and Karen, J. Shaw Aminoglycoside resistance genes aph (2)-Ib and aac-Im detected together in strains of both Escherichia coli and Enterococcus faecium. Antimicrobial agents and chemotherapy; 2001; Vol.45.PP. 2691- 2694. 31. MarcGalimand;PatriceCourvalin;andThierr yLambert.Plasmid-mediated high – level Iraqi J Pharm Sci, Vol.20(2) 2011 Combinations of antimicrobials against resistant E. coli 74 resistance toaminoglycosides in Enterobacteriaceae due to 16S rRNA methylation, antimicrobial agents and chemotherapy;2003;Vol. 47.PP.2565-2571. 32. Piddoch, LJ. Mechanisms of flouroquinolon resistance. Drugs;1999;58 (suppl2)11-18. 33. Sisson, G.; Goodwin, A.; Raudonikiene, A.; Hushes, N.J.; Mukhopadhyay ,A.K.; Berg, D.E.; and Hoffiman ,P.S.Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Antimicrobial agents and chemotherapy;2002; 46, 2116-212. 34. Minhchan Plac Ngugen; Paul Louis Woerther; Mathilde Bouvet; Antoine Andremon; Roland Leclercq; and Annuie Canu .Escherichia coli as reservoir for macrolide resistance genes.Emerging infectious disease;2009;Vol. 15. No.10. 35. Philippon, A.; Arlet, B.; Jacoby, GA. Plasmid –determined AmpC- type β- lactamases. Antimicrobial agent chemotherapy; 2002; 46:1-11.