Nova Explore Publications Nova Journal of Medical and Biological Sciences Vol. 3(1), 2014:1-5 PII: S2292793X1400003-3 www.novaexplore.com Multidrug resistance of Enterobacter Aerogenes isolated from bovine animals in Okada, Edo state, Nigeria Christiana Jesumirhewe 1*, Donald Arinzechukwu Umebuani 1, Peter Oladejo Ogunlowo 1 Introduction Enterobacter aerogenes is widely distributed in the soil, water, dairy products and in the intestine of animals as well as humans. They are most frequently found in the gastrointestinal tract [1] Antimicrobial resistance has been found in pathogenic and non-pathogenic strains of E. aerogenes which could be acquired from other organisms and can also be multidrug resistant [2]. Microbial resistances to antibiotics are on the rise because of inappropriate use of antibiotics in human medicine and also because of practices in the agricultural industry [3]. Multidrug resistance in an organism is the resistance of the microorganism to two or more classes of antimicrobials used against it [4]. E.aerogenes is resistant to most penicillins and cephalosporins because of the production of chromosomal beta lactamase with cephalosporinase activity. Additionally many are resistant to tetracycline, chloramphenicol, streptomycin as well as other aminoglycosides and flouroquinolones [5]. The most widely used types of antimicrobials in food animal production are the older classes of antibiotics: tetracycline, sulfonamide, trimethoprim, and β-lactams [6]. Factors involved in the emergence of antibiotic resistance in farm animals is primarily through inconsistent administration of antibiotics that exert selective pressure when feeding human antibiotics to farm animals to enhance their growth or treat infection [7]. Kang et al., 2005 [8] showed that the use of antimicrobial drugs in food animals is strongly associated with antibiotic resistance in animals. Through the food chain, antibiotic resistant animal or bacteria can be transferred to humans [9]. Antibiotic resistance in microbes leads to severe consequences. Infections caused by resistant microbes fail to respond to treatment resulting in prolonged illness and greater risk of death, longer hospitalization and infection periods which increases the number of Nova Journal of Medical and Biological Sciences Page: 1
infected people in the community [10]. Enterobacter strains efficiently exchange genetic material with pathogens such as Salmonella, Shigella, Yersinia and Vibro species and such resistance factors could be transferred and lead to development of multidrug resistance [11]. The aim of this study was to describe multidrug resistance among Enterobacter strains seen in bovine animals in Okada Edo state, Nigeria and the need for it to be regularly monitored especially in developing countries as it could be transferred to humans which could result in serious health implications. Methods A total of twenty two isolates of Enterobacter aerogenes obtained from fifty feacal sample of healthy bovine animals collected between March-July 2011 were included in this study. Identification of strains was confirmed [12]. Antimicrobial susceptibility test was performed with all isolates by the disc diffusion method based on performance standards for antimicrobial susceptibility testing by the Clinical and laboratory Standard Institute [13] against different antimicrobials including those commonly used in bovine animals in Nigeria. The antibiotic concentration per disc was as follow: Sulfamethoxazole/ trimethoprim (25µg), chloramphenicol (30µg), sparfloxacin (5µg), ciprofloxacin (5µg), amoxicillin (30µg), amoxicillin/clavulanic acid (30µg), gentamicin (10µg), perfloxain (5µg), ofloxacin (5µg), streptomycin (5µg), ceftriaxone (30µg), ceftazidime (30µg), cefotaxime (30µg), cefepime (30µg), imipenem (10µg). Multidrug resistant strains were defined as resistance to two or more classes of antimicrobials tested [4] Organisms shown to be multidrug resistant were selected as possible ESBL producing multidrug resistant isolates by the double disc synergy test method using ceftazidime, cefotaxime and ceftriaxone discs (30µg each) with amoxicillin-clavulanic acid (20/10µg) placed towards the centre of the agar plate [14]. The minimum inhibitory concentration of four antibiotics representing members of three classes of antibiotics which included ceftriaxone, cefepime, gentamicin and ciprofloxacin against each of the extended spectrum beta lactamase producing multidrug resistant isolates in this study were determined by the broth dilution method [15]. Results The cultural and biochemical characteristics of the bacterial isolates produced results that agreed with their identities. The overall rates of resistance for the 22 E.aerogenes isolates analyzed are provided in Fig 1. Among the antibiotics tested, imipenem (0%), gentamicin and streptomycin (13.6%) demonstrated the lowest rate of resistance and cefotaxime (100%) demonstrated the highest. Generally the resistance rates to the antibiotics were high in this study. All the isolates were found resistant to at least a class of antibiotics except the aminoglycosides. Among the 22 isolates tested, 4.55% were susceptible to all agents studied (Fig 2) and 13.6% were resistant to a single agent. Multidrug resistant isolates accounted for 81.8% (18) of the 22 isolates as isolates resistant to two or more classes of the antibiotics. Resistance was predominant among the folate pathway inhibitors represented by trimethoprim-sulfamethoxazole (SXT) in this study. All the antimicrobial resistant phenotypes that were identified among the multidrug resistant isolates were listed in Table 1. Remarkably all multidrug resistant isolates had distinct antimicrobial phenotypes. Out of the eighteen multidrug resistant E.aerogenes bovine isolates tested for ESBLs seven isolates were found to be ESBLs producers while the remaining isolates were non-producers. The various minimum inhibitory concentrations (MIC) values against the multidrug resistant isolates that are ESBL producers are given in Table 4. Generally, the isolates showed high level of resistance to all the antibiotics tested with high MICs. Discussion Enterobacter species can be found in the faeces of humans, animals, water, plant materials, insects and dairy products [16]. Reports of multidrug resistance and a high incidence of extended spectrum beta lactamase production among human clinical Enterobacter isolates have been recounted in our environment [17]. Previous reports on multidrug resistant Enterobacter aerogenes that are bovine isolates are scarce especially in developing countries like Nigeria. Fig 1: Antimicrobial susceptibility results for 22 E.aerogenes isolates from bovine animals Nova Journal of Medical and Biological Sciences Page: 2
*=81.8% (18 of 22) isolates were resistant to two or more classes of antibiotics and defined as multidrug resistant Fig 2: Resistance to one or more classes of antibiotics among 22 isolates of E.aerogenes from bovine animals tested against seven different classes of antibiotics Table 1: Antimicrobial Resistant Phenotypes in Multidrug resistant Enterobacter aerogenes in the Bovine Isolates Enterobacter aerogenes Isolates Antimicrobial resistance phenotypes 2 SP,AUG,CN,CRO,CAZ,CTX,FEP 4 SXT,SP,CPX,AM,AUG,CRO,CAZ,CTX,FEP 5 SXT,SP,AM,AUG,CRO,CAZ,CTX 6 SP,CPX,AM,CRO,CAZ,CTX,FEP 7 SXT,CPX,CRO,CAZ,CTX,FEP 8 SXT,CH,SP,AM,OFX,CRO,CAZ,CTX,FEP 9 SXT,SP,CPX,AM,CRO,CAZ,CTX,FEP 10 SXT,SP,CPX,AM,AUG,CN,OFX,CRO,CAZ,CTX,FEP 11 SXT,SP,AUG,CRO,CAZ,CTX,FEP 12 SP,CPX,AM,OFX,CRO,CAZ,CTX,FEP 13 SXT,CH,SP,CPX,AM,OFX,S,CRO,CTX 14 SXT,CH,SP,CPX,CRO,CAZ,CTX,FEP 16 SXT,SP,CPX,AM,OFX,CRO,CAZ,CTX,FEP 17 SXT,SP,CPX,OFX,CRO,CAZ,CTX 18 SXT,CH,SP,CPX,AM,S,CRO,CAZ,CTX 20 SXT,CH,SP,CPX,CN,CRO,CAZ,CTX,FEP 21 SXT,SP,CPX,AM,AUG,S,CAZ,CTX 22 SXT,AM,CTX Key- SXT-Sulfamethoxazole/trimethoprim, CH-Chloramphenicol, SP- Sparfloxacin, CPX- Ciprofloxacin, AM- Amoxicillin, AUG- Amoxicillin/clavulanic acid, CN- Gentamicin, PEF- Perfloxacin, OFX- Ofloxacin, S- Streptomycin, CRO- Ceftriaxone, CTX- Cefotaxime, FEP- Cefepime Nova Journal of Medical and Biological Sciences Page: 3
Table 2: Minimum inhibitory concentrations (µg/ml) of selected antibiotics against the extended spectrum β-lactamase positive multidrug resistant isolates No of isolates Clinical Isolates CRO FEP GEN CPX 4 E.aerogenes 50 50 >50 >50 5 E.aerogenes 50 12.5 >50 3.13 6 E.aerogenes >50 50 >50 50 9 E.aerogenes >50 50 >50 >50 13 E.aerogenes 12.5 25 >50 6.25 20 E.aerogenes >50 50 >50 50 21 E.aerogenes 25 25 >50 >50 Key:CRO- Ceftriaxone, FEP-Cefepime, GEN-Gentamicin, CPX- Ciprofloxacin >50- At concentrations of 50 µg/ml of the antibiotic, there was visible growth (Minimum Inhibitory Concentration is greater than 50 µg/ml) The results in this study show that carbapenems are still the most effective agents against Enterobacteriaceae [18] which is consistent with our results. All multidrug resistant isolates had a distinct antimicrobial resistant phenotype. Our study showed multidrug resistance to β-lactam antibiotics, flouroquinolones, chloramphenicol, tetracycline and aminoglycosides among veterinary isolates although on a relatively small scale. The high rates of multidrug resistant and ESBL producing isolates found in this study are of significant interest. A possible explanation could be the use of some of these antibiotics in the animals. In Nigeria, extended spectrum cephalosporins are not in use in veterinary practice [19] however their resistant determinants could be selected by other drugs frequently used in food animals. Chah et al., 2007 [19] reported the wide use of ampicillin in poultry production in Nigeria which may provide a selective pressure favouring the emergence of E.coli strains that produce ESBL enzymes. These food animals may serve as a reservoir of ESBL producing strains which could be transferred to humans and other animals. Differences in antimicrobial usage, infection control practices and unrecognized factors may provide a selective pressure favouring the emergence of E.aerogenes strains that produce ESBL enzymes. Conclusion Results from this study show that bovine animals may therefore serve as reservoir of extended spectrum beta lactamase producing and multidrug resistant E.aerogenes which could be transferred to humans and other animals. There is an urgent need to recognize all factors involved in antimicrobial resistance, control the use of antimicrobials and select the best strategies to combat the development of resistance. References 1. Atlas RM, Bartha R. Microbial ecology: fundamentals and applications. 1986. 2. Oppegaard H, Steinum TM, Wasteson Y. Horizontal transfer of a multi-drug resistance plasmid between coliform bacteria of human and bovine origin in a farm environment. Applied and environmental microbiology. 2001;67:3732-4. 3. Khachatourians GG. Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. Canadian Medical Association Journal. 1998;159:1129-36. 4. Refkia H, Marlyn S. Advanced cystic fibrosis lung diseases in children: Pulm. Med; 2001. 448-53 p. 5. Greenwood D, Richard C, John P. Medical Microbiology: Edinburg Churchill Livingstone; 2002. 6. Fleming DM. The state of play in the battle against antimicrobial resistance: a general practitioner perspective. Journal of Antimicrobial Chemotherapy. 2007;60:i49-i52. 7. McEwen SA, Fedorka-Cray PJ. Antimicrobial use and resistance in animals. Clinical Infectious Diseases. 2002;34:S93-S106. 8. Kang HY, Jeong YS, Oh JY, Tae SH, Choi CH, Moon DC, et al. Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea. Journal of Antimicrobial Chemotherapy. 2005;55:639-44. 9. Organization WH. Use of antimicrobials outside human medicine and resultant antimicrobial resistance in humans. Geneva: World Health Organization. 2002. 10. Bisht R, Katiyar A, Singh R, Mittal P. Antibiotic resistance-a global issue of concern. Asian Journal of Pharmaceutical and Clinical Research. 2009;2:34-9. 11. Puente J, Finnely B. Pathogenic Escherichia coli. In: Principles of bacterial pathogenesis. San Diego: Academic press; 2001. 387-486 p. 12. Cheesbrough M. District laboratory practice in tropical countries: Cambridge university press; 2006. 13. Cockerill F, Wikler M, Bush K, Dudley M, Eliopoulos G, Hardy D. Clinical and Laboratory Standards Institute performance standards for antimicrobial susceptibility testing: Twentieth informational supplement M100-S20. Wayne, Pennsylvania: CLSI. 2010. 14. Thokar M. Extended spectrum beta lactamases: Physcians Academy; 2010. 26-7 p. Nova Journal of Medical and Biological Sciences Page: 4
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