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° North 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 identification of E. coli and Shiga toxin-producing E. coli O157:H7.
Microbiological procedures
Isolation of E. coli was attempted according to [37] with slight modification. 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 flame 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 identification for E. coli. Colony morphology and colors on MCA and EMB agar plates together with the Gram stain procedure were used as an initial identification 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 confirmation of identification [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 confirmed 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 amplification of the pal, eaeA stx1, and sxt2 virulence genes of E. coli
E. coli genomic DNA extraction and purification were performed as per the protocol given by PureLink® Genomic DNA Purification 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 modified by [51]. The amplification was done according to the protocol reported by [52] and [53] using the following specific primers: ECPAL-F-5’GGC AAT TGC GGC ATG TTC TTC C3’ and ECPAL-R-5’CCG CGT GAC CTT CTA CGG TGA3’ for E. coli (280 bp); EHEC-F-5’TGG TAC GGG TAA TGA AAA3’ and EHEC-R-5’AAT AGC CTG GTA GTC TTG T3’ for eae gene of EHEC (360 bp); Stx1-F-5’ATA AAT CGC CAT TCG TTG ACT AC3' and Stx1-R-5’AGA ACG CCC ACT GAG ATC ATC3' for stx1 (180 bp); and Stx2-F-5’GGC ACT GTC TGA AAC TGC TCC3' and Stx2-R-5’TCG CCA GTT 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 final 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 amplification 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 final 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 five well-isolated 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 table of the Clinical and Laboratory Standard Institute [56].
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 (χ2) test was applied to assess the association.