The study was carried out in Addis Ababa, the capital city of Ethiopia. The city covers 540 km² and is divided into 10 sub-cities (Figure 1). The city lies at an elevation of 2,355 m above sea level and is located at 9°1′48″N 38°44′24″E. The city has minimum, maximum, and average temperatures of 14, 21 and 17.5°C, respectively. The capital city has an estimated human population of 3.15 million.

A cross-sectional study was conducted from October 2018 to December 2019 to determine the prevalence and antimicrobial susceptibility pattern and multidrug-resistant profile of E. coli O157:H7 serotypes in retail raw beef samples obtained from butchery shops, in Addis Ababa, the capital city of Ethiopia.

The sample size required was calculated according to Thrusfield (21), from an expected pooled prevalence of 6.5 for the butcher shops (11, 12, 14–16) with a defined precision of 5% and a level of confidence of 95%.

where Z = z statistic for level of confidence; n = required sample size; P_exp = expected prevalence and a desired absolute precision (d) of 0.05, Z = 1.96. Therefore, the minimum sample sizes were 49 butcher shops. However, in order to increase the precision of the study, a total of 384 butcher shops were included.

The study samples were retail raw beef. A list of active and legally registered butcher shops within the 10 sub-cites and their distribution lines were obtained from Addis Ababa Abattoir Enterprise. A total of 384 butcher shops were selected using the simple random sampling method, and the butcher shops were visited only once A raw beef sample was purchased from each of the randomly selected butcher shops as it was sold to the consumer.

Each sample was placed in a sterile individual plastic bag. The sample was identified by its exclusive sample identification number, which was written on the plastic bag, alongside the sub-city and the date of sampling. Finally, the sample was transported to the Microbiology Laboratory of the Aklilu Lemma Institute of Pathobiology, Addis Ababa University, at cold temperature in a cool box. Upon arrival to the laboratory, the samples were stored in a refrigerator at ±4°C. The samples were processed within 6–12 h from arrival. The detection of E. coli O157:H7 was administered consistent with the protocol of ISO-16654:2001 standard (11).

Twenty-five grams of raw beef was weighed and cut into smaller pieces with a sterile scalpel blade on a sterile plate and put in a sterile Stomacher bag. Then, 225 ml of modified Tryptone Soya Broth (TSB) supplemented with Novobiocin (mTSB+N) (1:9) was added to the raw beef and homogenized (Stomacher 400; Seward Medical, Worthing, United Kingdom) at high speed for 2 min. The enrichment sample was then incubated aerobically at 41.5°C for 24 h.

All enriched broths were plated on to cefixime tellurite sorbitol MacConkey agar (CT-SMAC) (Oxoid, Basingstoke, England), supplemented with 0.05 mg/L cefixime and 2.5 mg/L tellurite (Oxoid, Basingstoke, England) (CT-SMAC) (Oxoid, Basingstoke, England) and incubated at 37°C for 24 h. After the incubation period, the CT-SMAC agar plates were examined for the presence of non-sorbitol fermenter colorless colonies, and subsequently, they were sub-cultured on Rainbow agar O157 (Hayward, Berkeley Heights, NJ, USA). The plates were then incubated for 20–24 h at 37°C and observed for the presence of typical black or gray coloration on Rainbow agar O157, indicating pure colonies (22).

Five typical colonies from each Rainbow agar O157 plate were sub-cultured on nutrient agar (Oxoid, Basingstoke, England) for biochemical confirmation by indole formation. The agar plates were incubated at 37°C for 18–24 h. One colony from the pure culture on nutrient agar was inoculated into a tube of tryptone/tryptophan medium (Oxoid, Basingstoke, England) and incubated at 37°C for 24 h. Then, 1 ml of Kovac's reagent (Oxoid, Basingstoke, England) was added and the tube allowed to stand at room temperature for 10 min. The formation of red color indicates a positive reaction (11).

Indole-positive colonies were examined for their serological reaction with antiserum to E. coli O157:H7 using RIM E. coli O157:H7 latex test (Oxoid, Basingstoke, England). Indole-positive colonies were sub-cultured from the nutrient agar to the sorbitol MacConkey agar (Oxoid, Basingstoke, England). For every isolate to be tested, one drop of test latex was dispensed into a well of the test slide. In like manner, one drop of E. coli control latex was dispensed into a separate well of the test slide. Using a plastic stick, a portion of the non-sorbitol fermenting colony (NSFC) was removed from the sorbitol MacConkey agar (SMAC) (Oxoid, Basingstoke, England) plate and emulsified in E. coli O157 test latex on the slide and spread over the reaction area. Using a fresh plastic stick, the process was repeated with the remaining NSFC and emulsified in E. coli control, latex on the slide. The slide was rotated using circular motions for up to 1 min or until agglutination appears. For E. coli O157 positives that agglutination occurs with the E. coli O157 test latex and the control latex is negative, the isolate was streaked from sorbitol MacConkey agar (Oxoid, Basingstoke, England) to a blood agar (Oxoid, Basingstoke, England) plate and incubated at 37°C for 18–24 h. After 18–24 h incubation, the sweep of growth from the blood agar plate was emulsified in a drop of E. coli H7 test latex. Colonies giving an agglutination reaction were confirmed as E. coil O157:H7 positive.

The antimicrobial susceptibility was performed, following the standard agar disk diffusion method consistent with CLSI (23) using commercial antimicrobial disks (Table 1). The antimicrobial agents were selected based on the use of antimicrobial agents in the ruminants, potential public health importance, and recommendations from the guideline of antimicrobial susceptibility testing from the Clinical and Laboratory Standards Institute (23).

Each isolated bacterial colony from pure fresh culture was transferred into a tube of 5 ml TSB (Oxoid, Basingstoke, England) and incubated at 37°C for 6 h. The turbidity of the culture broth was adjusted using sterile saline solution or added more colonies to get turbidity comparable with that of 0.5 McFarland standards. The diluted bacterial suspensions were swabbed in three directions uniformly on the surface of Mueller–Hinton agar plates using sterile cotton swabs. After the plates were dried (about 10 min), with the aid of sterile forceps, antibiotic-impregnated disks were placed to the surface of the inoculated plates. Then, the plates were incubated aerobically at 37°C for 24 h. Finally, the diameter of the inhibition zone formed around each disk was measured on a black surface using a transparent ruler by placing it over the plates. The results were classified as sensitive, intermediate, and resistant according to the CLSI (23). E. coli (ATCC 25922)-type strains were used as a positive control.

Multidrug resistance (MDR) was defined as a resistance of a bacterial strain for at least one agent in three or more antimicrobial categories (24).

The study protocol was ethically approved by the Institutional Review Board of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (Minutes Ref NO: ALIPB IRB/006/2011/2018).

The data were entered and coded in MS Excel and then analyzed using IBM SPSS version 25.0 (25). The prevalence was determined by dividing the number of positive samples by the total number of samples examined. Descriptive statistics such as frequency and percentages were used to describe the proportion of resistant, intermediate, or susceptible strains. The difference in prevalence by sub-city was determined using the chi-square (χ²) test. A p-value < 0.05 was considered indicative of a statistically significant difference.

Out of 384 raw beef samples examined, 14 (3.64%) (95% CI = 1.77–5.51%) were positive to E. coli O157:H7 serotypes.

E. coli O157:H7 serotypes were detected in Addis Ketema (2%), AkakiKality (1.47%), Arada (14.29%), Bole (4.65%), Gullele (7.14%), Kirkos (69%), KolfeKeraneo (0%), Lideta (6.9%), Nefassilk (1.72%), and Yeka (4.76%). Variation in the prevalence between the butcher shops from the different sub-cities was not statistically significant (p > 0.05) (Table 2).

The result of the antimicrobial susceptibility test of the14 E. coli O157:H7 serotypes isolated from raw beef samples with 13 selected antimicrobial agents is shown in Table 3.

All the 14 E. coli O157:H7 serotypes' isolates from raw beef were found to be susceptible to amikacin (100%), ciprofloxacin (100%), and ceftriaxone (100%). Furthermore, the isolates showed high susceptibility to sulfamethoxazole-trimethoprim (92.8%), nalidixic acid (92.8%), gentamicin (85.7%), cefoxitin (78.5%), kanamycin (71.4%), and amoxicillin-clavulanic acid (64.2%). The results of the present study on antimicrobial sensitivity test indicated high resistance to ampicillin (92.8%), nitrofurantoin (92.8), and tetracycline (50.0%).

Out of the 14 E. coli O157:H7 isolates, 8 (57.14%) were found to be resistant to three or more antimicrobial categories. MDR profiles against three, four, and five antimicrobial categories were resistant to 5 (35.7%), 1 (7.1%), and 2 (14.3%), respectively. The frequency of resistant phenotype was more common for ampicillin, nitrofurantoin, and tetracycline (Table 4).