Study design, area and period
Laboratory based cross- sectional descriptive study design was conducted in Jimma town, Southwest Ethiopia from March to June, 2016. Jimma town is found at 352Kms, Southwest of Addis Ababa, capital city of Ethiopia. The town is divided in to 17 administrative kebeles. According to the information obtained from Jimma town trade and industry office, the town has one municipality abattoir and around 120 meat retailer shops which directly receive slaughter service from the abattoir. Eighty eight (88) meat retailer shops were purposively selected from five most populated kebeles of the town (Mendera Kochi, Kosa Kito, Mentina, Hermata Merkato, and Bochobore) and the required samples were collected from those meat retailer shops operating during the study period in the five kebeles of the town.
Laboratory sample collection
A total of 168 samples (88 minced meats of cattle and 80 swabs) were collected from meat retailer shops that operating during the study period in the five kebeles of the town. We made two visits of meat retailer shops per week for consecutive 4 weeks and within this period of time a total of 168 samples were collected. Eighty eight raw minced meat samples were collected from meat displayed for vending at the butcher shops. Twenty five grams (25 g) of minced meat samples were collected in sterile, separated plastic bags and labeled with permanent marker. The remaining 80 swab samples were also collected from hands of meat handlers, protective clothing, knives and chopping boards of the butcher shops at the beginning of operation. Swab samples were taken from 15 to 20 cm2 of the hands of meat handlers and protective clothing and from the surface of meat-cutting equipment such as knives and wooden chopping boards using sterile, buffered peptone water (BPW) moisten cotton swabs. The collected swab samples were returned into a separate test tube containing 9 ml sterile BPW, and all the collected samples were labeled, packaged in sterile plastic bags and carried to the microbiology laboratory of Jimma University in a cold box within 2 hrs of collection for processing .
Minced meat: is defined as a boneless meat that has been reduced to fragments .
Sanitary status of butcher shops and hygienic practice of meat handlers
Sanitary status of the butcher shops and hygienic meat handling practices of workers were assessed by the use of observational checklist at Jimma town. Two visits of butcher shops were conducted per week for consecutive 4 weeks and the following variables were used: availability of clean cold and warm tap water, availability disinfectant and soap, regular hand washing practice during work, disinfection of the floor and processing tools before and during the work, protective clothing is clean, use of separate and washable chopping boards and knives for processing of abdominal organs and other parts of meat, whether the entire process was done in the same area without separation, whether the same buckets of water were used for cleaning knives, washing hands, whether butcher shops’ floor is constructed of concrete ceramic and has no crack, dust, rodent and insects and whether the meat displayed for vending protected from dust and flies.
Isolation and identification process
A 25-g of minced meat of cattle were weighted and suspended into appropriate sterile cylinder/beakers that containing about 225 ml 0.1% buffered peptone water (BPW) and homogenized by shaking for 5 minutes in a sterile stomacher and incubated at 37 °C for 24 h for enrichment purposes. Similarly, all the collected swab samples were suspended into a test tube containing 9 ml sterile buffered peptone water (BPW) immediately at the collection sites and incubated at 37 °C for 24 h. After gently mixing, one loop-full (10 μl) of the overnight culture was streaked onto a MacConkey agar (Oxoid, UK) and incubated for 24 h at 37 °C .
Identification of the isolates
The isolated colonies were first screened by their colony morphology, pink colour producing lactose fermenting colonies, gram staining techniques and further identified as E. coli by motility and other relevant biochemical tests, such as indole, citrate, urease, methyl red, gas and acid production tests. All the confirmed isolates were refrigerated at 2-8 °C until antimicrobial sensitivity test and ESBLs-production test was done.
Screening of ESBL-producing isolates
The ESBL screening test was performed by standard disk diffusion method by using ceftazidime (30 μg) and cefotaxime (30 μg) disks. After adjusting 0.5 McFarland’s standard as indicated above, the suspension was inoculated onto Muller-Hinton agar (Oxoid, UK) with sterile cotton swab, and then the above two antibiotics disks were placed on the inoculated plate and incubated at 37 °C for 24 h. Isolates with reduced susceptibility to cefotaxime (≤ 27 mm) and ceftazidime (≤ 22 mm) around the disks were suspected as ESBLs- producers as recommended by CLSI guidelines .
Conformation of ESBLs- producing isolates
Those isolates with reduced susceptibility to cefotaxime (≤ 27 mm) and ceftazidime (≤ 22 mm) were conformed for ESBLs- production using double disk approximation or double disk synergy (DDS) method. After inoculation of the suspension onto Muller-Hinton agar (MHA), a disk of amoxicillin + clavulanic acid (20/10 μg) was placed in center of the plate and then the disks of cefotaxime (30 μg) and ceftazidime (30 μg) were placed at a distance of 20 mm from the central disk on the same plate . The plate was incubated at 37 °C for 24 h and examined for an enhancement of inhibition zone of the β-lactam drugs caused by the synergy of the clavulanate in the amoxicillin- clavulanate disk was interpreted as positive for ESBLs-production.
Antibiotic sensitivity testing
The antibiotic sensitivity testing was performed by using Kirby-Bauer disc diffusion technique. The suspension of the growth were inoculated onto Muller-Hinton agar and cefotaxime (30 μg), Ceftriaxone (30 μg), ceftazidime (30 μg), amoxicillin-clavulanic acid (20/10 μg), penicillin (10 μg), ampicillin (10 μg), gentamycin (10 μg), amikacin (30 μg), neomycin (10 μg), ciprofloxacin (5 μg), erythromycin (15 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), and tetracycline (30 μg) were placed on to the plate using sterile forceps. After overnight incubation of the plate at 37 °C, the zone of inhibition was measured by using sliding calipers and interpreted by comparing zone of inhibition with Kirby – Bauer chart as recommended by CLSI guidelines . K. pneumonia ATCC* 700603 and E. coli ATCC*25922 were used as positive and negative control strains to monitor the quality of susceptibility testing and ESBLs detection methods. Multi drug resistance (MDR) is defined as a resistance to at least one agent in three or more antimicrobial classes .
The data was analyzed by using SPSS version 16.0 with an emphasis on the human health significance of the findings. The difference in resistance between ESBL-producer and non-ESBL- producing strains were analyzed by using chi-square (X2) testing and p-value < 0.05 were regarded as statistically significant.