Rate of induction of resistance in Fecal E-coli against cefquinome as compare to ceftriaxone after continuous passage in vivo

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1 10-15 Rate of induction of resistance in Fecal E-coli against cefquinome as compare to ceftriaxone after continuous passage in vivo Ahmed H. Mohammed* Ali A. Al-Khayyat** Luna Ahmed Kafi** * College of Medicine, University of ThiQar ** College of Veterinary Medicine, University of Baghdad, Iraq ahmed_hamza @yahoo.com Astract: The study was conducted to evaluate the development of resistance for fecal E. coli against third and fourth generations of cephalosporins. This acteria was chosen ecause they represent the pool of resistance elements that are availale for transfer to other acterial species including pathogens.three groups of 5 (mice) were used µg/Kg) of ceftriaxon and(12.5µg/kg) of cefquinome, were given a su therapeutic dose orally for the first and second groups respectively. While the third group were given distilled water only as a control. Then isolation, purification and identification of fecal E-coli from GIT was done. Morphological and iochemical tests had een used to make sure that isolated acteria was E.coli. It has een oserved that the mean value minimum inhiitory concentration (MIC) of the isolated acteria from oth of first and second groups was compared with the control group.mic of oth antiacterials in comparison with the control group which were 1.18, 1.37µg/ml for ceftriaxone,cefquinome respectively. While the MIC values for ceftriaxone and cefquinome were and 4.6 µg/ml respectively.this means that the significancy (P<0.05) was folds and 3.35 folds in the third and fourth generation generations respectively in comparison with the control of each antiacterial.we concluded that antiiotic resistance may not e only a consequence in pathogenic acteria ut also in normal flora which could contriute this resistance to other microorganism. Key word :MIC :Minimum Inhiition Concentration, PBPs: penicillin-inding proteins معذل استحذاث انمقاومة في االشيريشيا انقونونية انبرازيه ضذ انسفكوينوم مقارنة مع انسفترياكسون بعذ تكرار تمريرها داخم انجسم انحي اح ذ ح ضة يح ذ* عه عض ض انخ بط** نب أح ذ كبف انعب ** *كه ت انطب, جبيعت ر قبس ** كه انطب انب طش, جبيعت بغذاد, انعشاق انخالصة: أجش ج ز انذساست نخ ق ى حط ش ان قب ي ت ف االش ش ش ب انق ن ت انبشاص, ف جسى انكبئ انح ضذ انج م انزبنذ انشابع ي انس فبن سب س انشبئعت االسخع بل ف اإل سب انح ا, قذ اخخ شث ز انبكخ ش ب نك ب ح زم يح ظ ي 10

2 ع بصش ان قب يت انخ حك يخبحت إن أ اع أخش ي انبكخ ش ب بض ب ان شض ت. أخزث ف انبذا ت رالد يجبي ع )فئشا ( كم يج عت ححخ عه خ س أفشاد. أعط ج )89.25 يكغى/يم( ي انسفخش بكض )12.5 يكغى/يم ) ي سفك و كشبع انجشع انعالج يش احذ ي ب ن ذ أسب ع, نه ج عت انال ن انزب عه انخ ان, حى اخز ان ج عت انزبنزت ك ج عت س طشة إعطبئ ب يبء يقطش فقظ ن فس ان ذة ان زك سة أعال, بعذ ا خ بء انفخشة ان حذدة حى عضل االش شش ب انق ن ت انبشاص بعذ ب حى إعبدة حأك ذ ت انجشر يت بعذ ب حى ححذ ذ ق انخشك ض ان زبظ األد MIC نهبكخ ش ب نكم يج عت )األ ن انزب ت( يقبس خ ب يع يج عت انس طشة. كب ج انق ى MIC نه ضبداث انح ت ن ج عت انس طشة 1.18 يكغى/يم نهسفخش بكض 1.37 يكغى/يم نهسفك و ب فس انطش قت حى ححذ ذ ق ى MIC نه ج عخ اال ن انزب ت بعذ اسخحذاد ان قب يت ن ب كب ج ق ى , MIC يكغى/يم نهسفخش بكض انس فك و عه انخ ان زا ع بأ اال ض ان سخ )0.05>P( كب ج ضعفب 3.35 ضعفب نهج م انزبنذ انشابع عه انخ ان ببن قبس ت يع يج عت انس طشة نكم يضبد ح. نقذ اسخ خج ي ز انذساست أ يقب يت ان ضبداث انح ت قذ ال حك فقظ نهبكخ ش ب ان سببت نأليشاض نك أ ضب ححصم نهفه سا انطب ع ت انخ ك أ حس ى ف فم ز ان قب يت إن انكبئ بث انح ت انذق قت األخش. Introduction: The increase in the numer of resistant and multiresistant strains of acteria is a major concern of health officials worldwide, particularly with the decline in the numer of new antiiotics availale for treatment. While much effort has een directed toward management and monitoring of antiiotic use and the prevalence of acterial within communities ( 1,2). Furthermore, antimicroial-resistant is spreading etween different acterial strains in different haitats has een demonstrated (3). The normal flora contains antiiotic resistance genes to various degrees, even in individuals with no history of exposure to commercially prepared antiiotics. Several factors seem to increase the numer of antiiotic-resistant acteria in feces. One important factor is the exposure of the intestinal flora to antiacterial drugs. Antiiotics used as feed additives seem to play an important role in the development of antiiotic resistance in normal flora acteria., (4). The knowledge of prevalence and patterns of antimicroial resistance in generic Escherichia coli might indicate the pool of resistance elements that are availale for transfer to other acterial species including pathogens(5). Generic E. coli are used to monitor changes in prevalence and patterns of resistance as these commensal acteria are regularly found in the gastrointestinal tract of animals and humans (6). Ceftriaxone a third generation cephalosporins P, N and C, 7-amino cephalosporinic acid, and with the addition of side chain, it ecame possile to produce semisynthetic compound,ceftriaxone is injectale cephalosporin agent,in addition.ceftriaxone are highly resistant to staphylococcal β- lactamases producers and has a good activity against Gram positive and Gram negative acteria (7).it is considered to e the drugs of choice for many infections caused y memers of the Enteroacteraciae (8). Ceftriaxone, a etalactam antiiotic is mainly actericidal. It inhiits the third and final stage of acterial cell wall synthesis y preferentially inding to specific penicillin-inding proteins (PBPs) that are located inside the acterial cell wall. Cefquinome is an aminothiazolyl cephalosporin extended spectrum etalactam and a memer of the fourth generation cephalosporins, Cefquinome has een used only in veterinary medicine and only for individual treatment, and is safe and well tolerated. Formulations for the US will e availale for parenteral injection, either as multiple or as singledose products.(9). it is a zwitter-ionic compound with improved penetration into the periplasmatic space of Gram-negative acilli and enhanced inding to penicillininding proteins.aim of our study that is isolation and identification of fecal E-coli 11

3 from mice and determine the sensitivity y measuring the (MIC) against cefquinome and ceftriaxone and determination possiility of induction of resistance of these microorganisms against the aove mentioned drugs in vivo y continuous treatment with su therapeutic doses(1/4) in mice. Materials and Methods: To determine the rate of antiiotic resistance of fecal E. coli from healthy mice and to infer it is induced y antiiotic use y giving orally su therapeutic dose, In the present studies white Swiss BALB/C mice were employed as the test animals,15 mice were taken and divided three groups respectively as following: 1- group one, 5 mice were given a su therapeutic dose orally (89.25µg/Kg) of ceftriaxon, once daily for one week. 2- Group two 5 mice were given su therapeutic dose orally (12.5µg/Kg)for cefquinome, giving once daily for one week (10) 3- Group three served are control five mice were given distilled water only for the same period. At the end of 7 days period, isolation, purification and identification of fecal E-coli from GIT was done y Fresh specimens of faeces (0.5 g) from 6 normal healthy mice were collected in sterile universal containers. They were transported quickly to the laoratory and processed within an hour of collection, cultured first on the rain heart infusion roth, incuated at 37, for 24hrs, transfer to macconkey agar secondly, and transfer to E.M.B agar. The diagnosis depends on the color, shape, size of colonies, also the confirmatory tests had een completed to make sure that isolates acteria was E.coli after that determined the mean MIC to acteria for each group (first and second) which compare with control group. Results and Discussion: Induction resistance in vivo y giving antiiotic orally at su therapeutic dose(1/4) for one week. An interesting oservation in this study is the induction of resistance to third and fourth generation cephalosporins in vivo. In an experiment designed to induce resistance in vivo for fecal E. coli to cetriaxone and cefquinome, after exposure of susceptile microorganisms to su therapeutic dose (1/4 ) of each of the two drugs for seven days.then isolation, purification and identification of fecal E- coli from GIT was done. Morphological and iochemical tests had een used to make sure that isolated acteria was E.coli., The results of MIC of the two antiacterials for control group (after giving distilled water only) estimated y tue dilution method are summarized in tale 1.These were 1.18, 1.37µg/ml for ceftriaxone, cefquinome respectively. This results showed that no significant difference in value of MIC with each drug ecause that third and fourth generation have een found to e effective against most major pathogens, including E. coli. The same process after giving drugs orally (su therapeutic dose) for induction of resistance the MIC values were set out in tales 2,3 and 4 they were and 4.6 µg/ml for ceftriaxone and cefquinome respectively, which mean an increase of 13.55, 3.35 folds respectively. The third generation showed significantly highest increase (13.55) folds, while the fourth generation showed the lowest increase (3.35) folds.these findings are in agreement with (11) found reduction in the proportion of sensitive coliforms excreted in pigs after chlortetracycline fed at concentrations of 10 or 20 grams per ton. (12), found in their study when used clindamycin, in short-term studies, cause disturances in the composition of the gut microiota as well as to select for resistance. These studies have mainly een ased on data from isolates and have 12

4 indicated a normalization of the flora a few weeks following withdrawal of the treatment However, y using molecular approaches focused on the genus Bacteroides, it is found longterm shifts in the composition of the intestinal microiota of individual sujects after a short-term administration of clindamycin (13). As mentioned efore exposure of microorganism to different levels of antiacterial drug may result in increase in degree of resistance as reported efore y many workers. (14) reported the use of road spectrum antiiotics creates a selective pressure on the acterial flora, thus increasing the emergence of multiresistant acteria, which results in a vicious circle of treatments and emergence of new antiiotic resistant acteria. The gastrointestinal tract is a massive reservoir of acteria with a potential for oth receiving and transferring antiiotic resistance genes, in the context that the reason for the development of resistance in vivo is due to replacement of sensitive serotypes y other already resistant serotypes. At the same time, it is possile to monitor the acquisition and persistence of resistance genes in the community. Together this information should help to provide knowledge of the natural dynamics of the normal microiota and help us to understand the long-term consequences of antimicroial treatment. This information is of great importance for the implementation of rational administration guidelines for antiiotic therapies. Tale 1: the result value of MIC of two antiacterials after giving distal water only for one week No of mice Value of MIC µg/ml for ceftriaxone Value of MIC µg/ml for cefquinome Mice Mice Mice Mice Mice * Mean ± stander error 1.18 ± ± 0.30 *Numer represent mean ± stander error P>0.05 Tale 2. show initial and final values of MIC of the ceftriaxone, after giving orally, contain su therapeutic dose (1/4 therapeutic dose) for one week. Initial MIC µg/ml final MIC µg/ml (giving No of mice (giving D.W only ) su therapeutic dose ) Mice Mice 2 Mice Mice Mice ± ± 2.44 * Mean ± stander error a *Numer represent mean ± stander error Different small letters mean significant (P<0.05) result etween concentration within group. 13

5 Tale 3. show initial and final values of MIC of the cefquinome, after giving orally, contain su therapeutic dose (1/4 therapeutic dose) for one week. No of mice Initial MIC µg/ml (giving D.W only ) final MIC µg/ml (giving su therapeutic dose) Mice Mice Mice Mice Mice ± ± 0.98 * Mean ± stander error a *Numer represent mean ± stander error Different small letters mean significant (P<0.05) result etween concentration within group. Tale.(4-13) show values of MIC of the control group and two antiiotics in vivo,after giving su therapeutic dose for one week. ANTIBIOTICS Value of MIC µg/ml of control groups (giving D.W only ) Value of MIC after giving su therapeutic dose for one week Folds of elevation Ceftriaxone 1.18 ± ± 2.44 Aa Cefquinome 1.37± ± 0.98 B 3.35 Numer represent mean ± stander error. Different small letters mean significant (P<0.05) result etween within group. Different capital letters mean significant (P<0.05) result etwee different groups. L.S.D: 2.9. References: 1. Costanzo, S.D. ; Mury, J. and Bates, J. (2005). Ecosystem response to antiiotics entering the aquatic environment, Marine Pollution Bulletin. 51: Batt, A.L. ; Bruce, I.B. and Aga, D.S., (2006). Evaluating the vulneraility of surface waters to antiiotic contamination from varying waste water 14 treatment plant discharges. Environmental Pollution. 142: Svarm, (2006). Swedish veterinary antimicroial resistance monitoring. The National Veterinary Institute (SVA), Uppsala, Sweden. : Henning, S. and Marianne, S.(2001). Review article of the Resistance

6 to antiiotics in the normal flora of animals. Vet. Res. 32, : Sunde, M. and Sorum, H. (2001). Self-transmissile multi-drug resistance plasmids in Escherichia coli of the normal intestinal flora of healthy swine. Micro. Drug Resist. 7 (2): Kim, S. ; Hu, J. ; Gautom, R. Kim, J. ; Lee, B. and Boyle, DS.(2007). CTX-M extended- spectrum eta-lactamases, Washington State. Emerg Infect Dis. 13: Neu, H. C. ; Aswapokee, N. ; Aswapokee, P. and Fu, K.P. (1979). HR576, a new cephalosporins active against Gram positive and Gram negative aeroic and anaeroic acteira. Antimicro. Agents Chemother. 15: Luke, C. H. (2011). Studies on Salmonella enterica and Escherichia coli with a focus on ceftiofur and the genetic resistance determinant lacmy-2. PhD Dissertation. Graduate Program in Veterinary Preventive Medicine,Ohio State University, US. 9. FDA/CVM(Food and Drug Administration Center for Veterinary Medicine) Guidance. (2006). Cefquinome formulations for parenteral injection for the treatment of ovine respiratory disease, : Michael, L. ; Dieter, I ; Norert, K. ; Astrid, M. ; Karl, S. ; Gerhard, S. and Elmar, S.(1991). Antiacterial Activities In Vitro and In Vivo and Pharmacokinetics of Cefquinome (HR IIIV), a New Broad- Spectrum Cephalosporin. Antimicroial agents and Chemotherapy, Vol. 35, No. 1; : Finlayson,M. and Barnum, D. A.(1973). The Effect of Chlortetracycline Feed Additive on the Antiiotic Resistance of Fecal Coliforms of Weaned Pigs Sujected to Experimental Salmonella Infection. Can. J. comp. Med. Vol. 37: Sullivan, A. ; Edlund, C. and Nord, C. E. (2001). Effect of antimicroial agents on the ecological alance of human microflora. Lancet Infect Dis 1, Jernerg, C.; Lo fmark, S.; Edlund, C. and Jansson, J. K. (2007). Longtermecological impacts of antiiotic administration on the human intestinal microiota. ISME J 1, Susanne, S. and Karen, A. K.(2011). Assessment of Bacterial Antiiotic Resistance Transfer in the Gut. Review Article, International Journal of Microiology; Volume 2011, Article ID :