Isolation, Identication, and Antimicrobial Susceptibility Pattern of Shiga toxin-producing Escherichia Coli O157:H7 from Food of Bovine Origin in Mekelle, Tigray, Ethiopia

Background: Escherichia coli O157:H7 is an emerging and major zoonotic foodborne pathogen with an increasing concern for the emergence and spread of antimicrobial-resistant strains which may results in sporadic cases to serious outbreaks in the whole world. Cattle have been identied as a major reservoir of the pathogen. This study aimed to isolate and characterize Shiga toxin-producing E. coli O157:H7 from raw milk, yogurt, and meat of bovine origin and determine their antimicrobial susceptibility pattern. Methods: A cross-sectional study was conducted from December 2014-June 2015 and a total of 284 milk and meat were collected from different sources in Mekelle. The collected samples were analyzed for the presence of E. coli and Shiga toxin-producing E. coli O157:H7 and determination of their antimicrobial susceptibility pattern following the standard bacteriological and molecular techniques and procedures, and antimicrobial sensitivity test. Results: Out of the total 284 samples, 70(24.64%) were positive bacteriologically to E. coli and 14.29% were found to be Shiga toxin-producing E. coli O157:H7. All (100%) E. coli isolates carried the pal and 41.67% eae gene (EHEC). Of these EHEC isolates 40% and 60% were positive for stx1 and stx2, respectively. E. coli isolates were showed the highest level of sensitivity for Gentamycin (91.7%) but the highest level of resistance to Amoxicillin (95.8%). Of the tested isolates, 18(75%) of E. coli showed multiple antimicrobial resistance. Conclusions: The current study revealed the occurrence of Shiga toxin-producing E. coli O157:H7 in foods of bovine origin in the study area. So, there is a chance of acquiring infection via the consumption of raw or undercooked food of bovine origin. Thus, awareness creation should be made on foodborne disease caused by Shiga toxin-producing E. coli O157:H7 with due consideration on the safe handling and consumption of food of animal origin.


Background
In Ethiopia, both food shortage and lack of appropriate food safety assurance systems are problems that have become obstacles to the country's economic development and public health safety [1,2]. Animal products are generally regarded as high-risk commodities in respect of pathogen contents, natural toxins, and other possible contaminants and adulterants [3]. Foodborne microorganisms are major pathogens affecting food safety and cause human illness worldwide as a result of the consumption of foodstuff, mainly animal products contaminated with vegetative pathogens or their toxins. Most of these microbes have zoonotic importance resulting in a signi cant impact on both public health and economic sectors [4]. Moreover, the emergence of multidrug-resistant pathogens presents a global challenge for treating and preventing disease spread through zoonotic transmission [5].
Data regarding foodborne diseases are extremely scarce at a national level and a few studies conducted in different parts of the country showed the poor sanitary conditions of catering establishments and the presence of pathogenic organisms like Campylobacter, Salmonella, Staphylococcus aureus, Bacillus cereus, and Escherichia coli [6,7,8,9,10]. E. coli found in humans can be categorized on basis of genetic and clinical criteria into three main groups: commensal, pathogenic (enteric or diarrheagenic), and extraintestinal pathogenic E. coli (ExPEC) [11]. The typical diarrheagenic strains include: enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli [12,13]. The E. coli that cause enteric disease have been divided into pathotypes, based on their virulence factors and mechanisms by which they cause disease [11,14]. One of these pathotypes, called Shiga toxin-producing E. coli (STEC), refers to those strains of E. coli that produce at least 1 member of a class of potent cytotoxins called Shiga toxin. The STEC are also called verotoxin-producing E. coli (VTEC). The names Shiga toxin (Stx), derived from similarity to a cytotoxin produced by Shigella dysenteriae serotype 1 [15], and verotoxin (VT), based on cytotoxicity for Vero cells [16] are used interchangeably. Those STEC that cause hemorrhagic colitis and hemolytic uremic syndrome are called enterohemorrhagic E. coli (EHEC) [14,17]. The Shiga toxin-producing E. coli O157 is synonymous with E. coli O157:H7 [18,19]. Pathogenicity of E. coli O157:H7 is encoded by a variety of plasmid, bacteriophage, and chromosomal genes [20]. The key virulence factor for the subset of EHEC is the Shiga toxin (Stx) family which contains two subgroups: Stx1 and Stx2-that share approximately 55% amino acid homology [11]. The ability to produce Shiga toxin was acquired from a bacteriophage presumably directly or indirectly from Shigella [20].
STEC is recognized as an important cause of food-borne disease in humans and causes large outbreaks worldwide [21]. E. coli O157:H7 is the leading cause of hemorrhagic colitis, hemolytic-uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP) in man. These illnesses may lead to death due to improper absorption of nutrients and destruction of certain tissues in the target organs [22,23]. People of all ages are susceptible to infection with STEC. However, the young and the elderly are more susceptible and are more likely to develop more serious symptoms [24]. Domestic and wild animals are sources of EHEC O157:H7 but the major animal carriers are healthy domesticated ruminants, primarily cattle and to a lesser extent, sheep, and possibly goats. Fresh meat and raw milk are, nevertheless, considered as common vehicles for E. coli, particularly for the EHEC (O157:H7) strain. Contamination of meat usually occurs during animal slaughter, as a result of poor slaughter practices, abattoir hygiene, and animal handling practices [20,25].
Foods of bovine origin are frequently implicated in human outbreaks of Shiga toxin-producing Escherichia coli (STEC) O157 [26,27]. Shiga-toxigenic E. coli is transmitted by the fecal-oral route by either consumption of contaminated food or water, from direct contact with infected animals, or via person-to-person contact. Moreover, series of studies on the resistance of E. coli which were isolated from animals and humans have strongly suggested that those bacteria which are resistant to antimicrobials used in animals would also be resistant to antimicrobials used in humans [28,29,30]. In general, antimicrobial resistance is a global public health problem, and growing scienti c evidence indicates that it is negatively impacted by both human and animal antimicrobial usages [31]. Although antimicrobial therapy is not the primary tool for treating infections caused by STEC O157:H7, multidrug-resistant (MDR) STEC O157:H7 is a public health issue as those strains participate in a reservoir of resistance genes that could be easily exchanged between Enterobacteriaceae in the host and the environment [32]. But little is currently known about the molecular basis of multidrug resistance in STEC O157:H7 isolates of food origin [33]. So, it is of paramount importance to systematically investigate and characterized this recurring food-borne disease. Thus, the current study was conducted to isolate and characterize Shiga toxin-producing E. coli O157:H7 from raw milk, yogurt, and meat of bovine origin from different sources in the study area and to determine the antimicrobial susceptibility pattern of the isolates.

Study area
The study was conducted from December 2014 to June 2015 in Mekelle city. Mekelle is the capital city of Tigray Regional State, situated about 783 km North of Addis Ababa at 38.5° East longitude and 13.5°N orth latitude at an altitude of 2300 above sea level. The city has a total population of 215,546 [34], 308 cafeterias, 292 restaurants, 258 supermarkets, and an active urban-rural exchange of goods which has 30000 micro and small enterprises [35]. The weather condition is hot and dry. The mean annual rainfall of the area is 628.8 mm and an annual average temperature of 21ºC [36].

Study design and study population
A cross-sectional study on Shiga toxin-producing E. coli O157:H7 was conducted from December 2014 to June 2015 on raw milk, yogurt, and meat samples collected from different sources and parts of Mekelle City, Tigray, Ethiopia. The study populations comprised of purposively selected milking dairy cows and slaughtered cattle found in Mekelle City.

Sampling technique and sample collection
A total of 284 samples of bovine origin, comprised of raw milk (n=145), yogurt (n=48), and meat (n=91), were collected using a purposive random sampling technique. These samples were collected based on the willingness of the owners until the required sample size was achieved. Raw milk samples were aseptically collected directly from the teats of lactating cows (n=100), whole-sellers (n=17), cafeterias (n=28), and similarly the yogurt samples were collected from dairy farms (n=26) and cafeterias (n=22) using a sterile universal bottle. However, the raw meat samples were collected from abattoirs (n=55), butchery shops (n=16), and restaurants (n=20) during slaughtering and selling. Then, the sections of meat samples were aseptically removed and placed in separate sterile plastic bags to prevent spilling and cross-contamination. Both samples were transported to the Veterinary Microbiology Laboratory of College of Veterinary Sciences, Mekelle University using an icebox and stored at +4°C until the laboratory work was conducted.
Isolation and identi cation of E. coli and Shiga toxinproducing E. coli O157:H7.

Microbiological procedures
Isolation of E. coli was attempted according to [37] with slight modi cation. A part of each sample (10 ml or 10 g) was enriched in peptone water (HiMedia, India) (90 ml) and was incubated at 37°C for 24 h. Enriched samples were inoculated on MacConkey Agar (MCA) (HiMedia, India) by four ame technique, and plates were incubated at 37°C for 24 h. Pink-colored colonies were considered presumptive of E. coli. Gram staining was performed as per procedures described by [38] to determine the size, shape, and arrangement of bacteria. The organisms revealed gram-negative, pink-colored with rod-shaped appearance, and arranged in single or in pair were suspected as E. coli.
A single well-isolated colony was picked from MCA and streaked on Eosin Methylene Blue Agar (EMB) (HiMedia, India) and incubated at 37°C for 24 h. The characteristic green metallic sheen growth of colonies is a presumptive identi cation for E. coli. Colony morphology and colors on MCA and EMB agar plates together with the Gram stain procedure were used as an initial identi cation of E. coli colonies [39]. Such colonies were taken from EMB into nutrient broth and agar for further biochemical examination. Standard biochemical tests (Catalase test, Indole, Methyl red, Voges-Proskauer test, Nitrate reduction, Citrate utilization, and Urease production) were used as con rmation of identi cation [40,41,42,43,44,45,46]. Triple Sugar Iron test was performed according to [47]. Carbohydrate fermentation tests of the isolates were performed according to the method of [43]. Colonies, that were con rmed as E. coli, were further be subcultured onto MacConkey Agar with Sorbitol. A bacterial strain that was used as quality control organisms in this study was a standard strain of E. coli ATCC 25922.
PCR ampli cation of the pal, eaeA stx1, and sxt2 virulence genes of E. coli E. coli genomic DNA extraction and puri cation were performed as per the protocol given by PureLink® Genomic DNA Puri cation Kit, USA, for Gram-negative organisms, and the total genomic DNA was checked by running on 1.0% agarose gel.
All the presumptive isolates' DNA were subjected to multiplex PCR for analyzing for the presence of the pal [48], eae [49], stx1, and stx2 [50] genes, and further modi ed by [51]. The ampli cation was done according to the protocol reported by [52] and [53]  ATC TGA CAT TCT G3' for stx2 (255 bp). Each reaction mixture (50μl) consisted of 5μl of 10x reaction buffer (500mM KCl, 15mM MgCl2, 100mM Tris HCl pH 8.3, 0.1%w/v gelatin), 5μl of template DNA, 1μl of each primer (the primers were used at a nal concentration of 100 M), 3μl of 10mM dNTP mixture (at a concentration of 100μl each), and 1μl of Taq polymerase (3U/l). The remaining volume of the reaction mixture was nuclease-free water. The ampli cation was carried using a Tianlong PCR Thermocycler with thermal cycling conditions of an initial denaturation at 94°C for 6 min, followed by 35 cycles of denaturation at 94°C for 45s, annealing at 55°C for 30s, extension at 72°C for 45s, and with a nal extension at 72°C for 6 min. Finally, PCR products were separated in a horizontal equipment system by running on a 1.5% (w/v) agarose gel containing 0.5g/ml ethidium bromide for 55 min at 110 V using 1XTAE buffer (40 mM Tris, 1 mM EDTA, and 20 mM glacial acetic acid, pH 8.0). The amplicons were visualized under UV-light gel doc and their molecular weight was estimated by comparing them with 100 bp DNA molecular weight marker (Solis BioDyne, Tartu, Estonia) [54].

Antibiotic sensitivity testing
The isolates of E. coli were screened for in vitro antimicrobial susceptibility using the agar disk diffusion method described by [55]. For this, the following seven different antibiotic discs (Oxoid Ltd., Basingstoke, Hampshire, England) with their concentrations given in parentheses were used in the antibiograms: Erythromycin (E) (15µg), Gentamicin (CN) (10µg), Amoxicillin (Amx) (2µg), Doxycycline (Doxa) (30µg), Tetracycline (TE) (30µg), Penicillin (P) (10µg), and Sulfamethoxazole (SXT) (20µg). Four to ve wellisolated colonies from nutrient agar plates were transferred into tubes containing 5 ml of a normal saline solution until it achieved the 0.5 McFarland turbidity standards, and then a sterile cotton swab was dipped into the adjusted suspension and then spread evenly over the entire surface of the plate of Mueller-Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, England) to obtain uniform inoculums. The plates were then allowed to dry for 3 to 5 min. Antibiotic impregnated discs were then applied to the surface of the inoculated plates with sterile forceps. After 18 to 24 h of incubation at 37°C, the plates were examined and the clear zone (inhibition zones of bacterial growth around the antibiotic disc including the disc) diameter for individual antimicrobial agents was measured using a digital caliper and then translated into Sensitive (S), Intermediate (I), and Resistant (R) categories according to the interpretation

Data management and analysis
All collected data were entered into Microsoft Excel Sheet and analyzed through the SPSS Version 16.
Accordingly, descriptive statistics such as percentages and frequency distribution were used to determine the prevalence in the food items, and Chi-square (χ 2 ) test was applied to assess the association.

Results
The prevalence of E. coli and Shiga toxin-producing E. coli O157:H7 Out of the total 284 samples collected from the different sample sources, 70 (24.64%) were found to be positive for E. coli. The sample-based prevalences of meat, raw milk, and yogurt samples were 41 (45.05%), 18 (12.41%), and 11 (22.91%), respectively. There was a signi cant difference (p < 0.05) among the different sample types in the prevalence of E. coli (Table 1). Of the total isolated E. coli isolates, 14.29% were found to be Shiga toxin-producing E. coli O157:H7.  Antimicrobial susceptibility pro le of E. coli isolates The E. coli isolates isolated from meat, raw milk, and yogurt were showed the highest level of sensitivity for Gentamycin (91.7%). However, the highest level of resistance was observed against Amoxicillin (95.8%) ( Table 3). Multidrug resistance was detected in 75% of the isolates.  61%). The present study was also revealed that 20.8% of the isolates were showed resistance against Sulfamethoxazole which lower than the ndings of [93] (36%). In general, in this study, 75% of the isolates were developed multidrug resistance which was higher than the reports of [59] (46.0%) and [60] (66.3%) but was lower than the report of [74] (93.2%). Antimicrobial resistance may arise either spontaneously by selective pressure or due to antimicrobial misuse by humans or overuse in feeding or treatment of animals by farmers [94].

Conclusion And Recommendations
The present study revealed a relatively high occurrence of E. coli as well as Shiga toxin-producing E. coli O157:H7 in food of bovine origin in Mekelle City and isolates developed a high level of multi-drug resistance to the antimicrobials tested. Hence, foods of bovine origin can serve as a potential vehicle for transmitting E. coli, in particular, Shiga toxin-producing E. coli O157:H7, and its presence indicates a serious public health hazard and give a warning signal for the possible occurrence of a foodborne outbreak in humans through the consumption of raw or undercooked food items. As a result, both its presence and development of a multi-drug resistance should receive particular attention and is an alert for the concerned bodies. Therefore, a coordinated effort is needed to reduce or eliminate the risk posed by these pathogens at various stages in the food chains and on the appropriate use of antimicrobials both in veterinary and human treatment regimes. Moreover, awareness creation should be made on foodborne disease caused by Shiga toxin-producing E. coli O157:H7 with due consideration on the safe handling and consumption of food of animal origin and selection and safe use of antimicrobials. Availability of data and materials

Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.