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Molecular Determination of Antimicrobial Resistance In Messele et al. Ann Clin Microbiol Antimicrob (2017) 16:55 DOI 10.1186/s12941-017-0233-x Annals of Clinical Microbiology and Antimicrobials RESEARCH Open Access Molecular determination of antimicrobial resistance in Escherichia coli isolated from raw meat in Addis Ababa and Bishoftu, Ethiopia Yohannes Equar Messele1*, Reta Duguma Abdi2,3, Shimels Tikuye Yalew1, Desiye Tesfaye Tegegne1, Bezina Arega Emeru1 and Gebremeskel Mamu Werid1 Abstract Background: Consumption of meat contaminated by E. coli causes a serious illness and even death to afected individuals. Recently the emerging of antibiotic resistant foodborne E. coli poses serious public health risks worldwide. However, little is known about the antibiotic resistance profle of E. coli in Ethiopia. This study aimed to determine the prevalence and Antimicrobial resistance (AMR) status of E. coli isolated from diferent type of meat. Methods: Overall 292 samples were collected from December 2015 to April 2016 from slaughterhouses to deter- mine the prevalence and AMR of E. coli isolated from raw beef, mutton, chevon and chicken meat from Addis Ababa and Bishoftu, Ethiopia. The isolates were screened for AMR against commonly used antibiotics circulating in the Ethiopian market. Both phenotypic and genotypic approach were employed for AMR detection using disc difusion test and PCR respectively. Results: The prevalence of E. coli was 63 (21.6%), indicating one sample in every fve samples harbors E. coli. Among these, the highest E. coli isolates was observed in chicken meat samples (37.0%; 27), followed by mutton (23.3%; 17), chevon (20.6%; 15) and beef (5.5%; 4). Results of disk difusion test on the 63 isolates showed that only 4.8% of them were not resistance to all antimicrobials tested. Multiple drug resistance (resistance to 3 drugs) was 46.0%. Signif- cantly high resistance to ampicillin (71.4%) and tetracycline (47.6%) was observed. Identifcation≥ of genes associated with AMR was also done using PCR. The prevalence of E. coli isolates harboring resistance gene responsible for tetracy- cline (tet(A)), beta lactams (blaCMY) and sulphanamide (sulI) antibiotics were found 65.1, 65.1 and 54.0%, respectively. Twenty-fve out of the 63 (39.7% %) E. coli isolates have got antimicrobial resistance gene to three or more classes of drugs. The associations of antimicrobial resistance phenotypes and resistance genes was also determined. The detec- tion of resistance trait against tetracycline, sulphametazole and chloramphenicol measured either phenotypically or genotypically were high. Conclusions: The rising levels of resistance E. coli to multiple antimicrobial dictate the urgent need to regulate and monitor antimicrobial use in both animals and humans. Keywords: Antibiotic resistance, Escherichia coli, Meat, Ethiopia *Correspondence: [email protected] 1 Ethiopian Institute of Agricultural Research, National Agricultural Biotechnology Research Center, P.O.Box 31, Holeta, Ethiopia Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Messele et al. Ann Clin Microbiol Antimicrob (2017) 16:55 Page 2 of 9 Background Methods Meat and meat products serve as important source of Sample collection and isolation of E. coli proteins for humans. However, recently the emerging Te study was carried out from December 2015 to antibiotic resistant foodborne pathogens combined with April 2016 to collect meat sample from Addis Ababa the injudicious use of antibiotics in animals bears consid- city abattoir enterprise and Alema farm slaughter slab erate public health threats worldwide. Usually, meat and found in Bishoftu town, Ethiopia. A total of 292 meat meat products gets contaminated by pathogens during sample including beef meat (n = 73), sheep meat animal slaughter and food processing. Escherichia coli (E. (n = 73), goat meat (n = 73) were collected from Addis coli) is the frequently isolated foodborne pathogens from Ababa municipal abattoir and chicken meat (n = 73) meat and meat products. Meat can be contaminated by from Alema farm slaughter slab located in Bishoftu E. coli during animal slaughter due to unhygienic slaugh- city. Sample were collected by swabbing from carcass ter practices, through airborne, rodents, insects, and gluteal muscle and inserted into 10 ml peptone water other animals [1]. Consumption of meat contaminated by containing test tube. Te samples were taken follow- E. coli causes a serious illness and even death to afected ing the guidelines of the International Organization for individuals [2]. Standardization [10]. Te sample were transported to Recently published reports indicated that the E. coli National Agricultural Biotechnology Research Center strains isolated from contaminated meat and meat prod- in an ice box for further processing. Te E. coli isolates ucts have become resistant to commonly used antibiot- were identifed by using standard bacteriological meth- ics. Tis is mainly due to injudicious usage of antibiotics ods, comprising of colony structure determination, in both humans and animals [3]. Te wide spread and using diferent culture media and biochemical tests as imprudent use of antibiotics in food animals is thought previously indicated [11]. Isolates were preserved at to be accountable for the emergence and wider spreading −80 °C in a trypticase soy broth with 10% glycerol for of antimicrobial resistant (AMR) bacteria in humans [3, further analysis. 4]. In humans, positive selection for drug resistant bac- teria have also been reported in the normal microfora Antimicrobial susceptibility testing of exposed individuals or populations. Tis indicates Antibiotic sensitivity was determined by disc difusion that antibiotic resistance can be developed in both com- method according to the guidelines of the Clinical and mensal and pathogenic bacterial strains and can even Laboratory Standards Institute [12]. Bacterial suspen- be transferred to other bacterial strains, including other sion was prepared by adding 2–4 colonies to a 5 ml tube pathogenic and environmental bacteria [5]. containing 0.9% normal saline (NaCl), to achieve absorb- Consumption of contaminated and/or uncooked meat ance of 0.17–0.18 at wavelength of 600 nm (equivalent poses the risks of acquiring foodborne E. coli strains [6] to 0.5 McFarland standards) [13]. Te suspension was causing a serious public health concern. Tese bacterial spread onto Mueller–Hinton agar media (HIMEDIA, strains easily harbors antibiotic resistant genes from one India) using a sterile cotton swab, and the antibiotic disc another. Tis is because genes encoding AMR determi- (Oxoid, UK) were placed on the top of the agar plate. Te nants that are carried on mobile genetic elements such inoculated plates were incubated aerobically at 37 °C for as plasmids and transposons of some bacterial strains 18–24 h, after which all E. coli isolates were tested for could be transferred to other bacteria strains during con- susceptibility using the following antimicrobial discs and tact [7], causing a threat to cure acute infections in man their corresponding concentrations: tetracycline (30 μg/ and animals. In Ethiopia, a review done by Alemu et al. disk); streptomycin (10 μg/disk); chloramphenicol (30 μg/ [8] showed that several pathogen have established resist- disk), sulfamethoxazole–trimethoprim (25 μg/disk), ance against oxytetracycline drugs. Similarly, oxytetracy- gentamycin (10 μg/disk); ampicillin, (10 μg/disk); eryth- cline and penstrep are the main prescribed antibiotics in romycin (15 μg/disk); by the Kirby-Bauer disk difusion Ethiopia [8, 9]. So far, no study has been carried out to method [14]. identify the antibiotic resistance genes of Escherichia coli in Ethiopia. DNA extraction and PCR amplifcation of resistance genes In this paper, we explain the phenotypic and genotypic Bacterial strains were grown overnight in nutrient agar sources of MDR in Escherichia coli isolates recovered (HIMEDIA, India) at 37 °C. A loop full of the colonies from diferent type of meat samples taken from Addis was added to 100 μl of sterile water. Bacterial DNA was Ababa city abattoir and Alema farm poultry slaughter extracted by boiling a bacterial suspension in water. After slab, Ethiopia. Tis enquiry pursues to deliver useful evi- boiling the suspension for 13 min, the suspension were dence on the prevalence and AMR profle of E. coli iso- frozen for 5 min in ice and centrifuged at 14,000 rpm for lated from beef, mutton, chevon and chicken meat. 15 min to pellet the cell debris [15]. Te supernatant from Messele et al. Ann Clin Microbiol Antimicrob (2017) 16:55 Page 3 of 9 the centrifuged tubes was transferred to new 1.5 ml clean Statistical analysis plastic tube and used as a template for PCR amplifcation. Te prevalence of E. coli infection was quantifed and Te purifed DNA were detected by electrophoresis in compared among meat samples of diferent livestock spe- 1.5% agarose gel and then kept at −20 °C for further use. cies. Prevalence of AMR was quantifed along with resist- Primers for AMR genes such as streptomycin (aadA1), ance patterns. Te data were analyzed by using SPSS tetracycline [tet(A)], gentamicin [aac(3)-IV], sulfona- software version 20 and P value was calculated using Chi mides (sul1), beta-lactams (blaSHV, blaCMY), eryth- square and Fisher’s exact tests to determine any signif- romycin [ere(A)] and chloramphenicol (catA1, cmlA) cant correlation. P value less than 0.05 was considered were used from published article. Te specifc primer statistically signifcant. sequences (Bioneer, South Korea) and the estimated size of the amplifed products for diferent resistance gene Results coding regions are found in Table 1.
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