Study area and study design
The current cross-sectional study was conducted from October 2018 to April 2019 in Bahir Dar city (located 565 km north-northwest of Addis Ababa) in ready-to-eat raw beef retailers of butcheries. Geographically Bahir Dar is located between 11.29° to 11.38° North latitude and 37.23° to 37.36° East longitude. Its average elevation is estimated to be 1810 meter above sea level. The city’s mean annual temperature ranges from 7.1°C to 29.7°C, whereas annual mean temperature was 20.85°C [18].
Sample size determination and sampling procedures
In Bahir Dar city, about 137 licensed butcher shops were operating on meat and meat products and all the butcheries were included in the sampling procedure. The lists of all butcher shops were obtained from the health centers. The sample size were calculated by using Thrusfield formula for small sampling population[19] and a total of 101 retailers were selected based on simple random sampling.
Data collection
From randomly selected butcher shops, about 250 gram of ready-to-eat raw beef (Kurt) samples were collected in sterile stomacher plastic bags and kept in icebox containing ice. The collected samples were immediately taken to Bahir Dar University, Institute of Technology food microbiology laboratory for homogenization and the homogenate were transported to the Amhara Public Health Institute (APHI) microbiology laboratory unit within 4 hours by keeping the cold chain for bacteriological analysis.
Bacteriological investigation
Isolation and identification of S. aureus from ready-to-eat raw beef was done according the methods described by ISO 6888-3 [20] and [21]. Briefly 25 gram of raw beef sample was transferred aseptically into a sterile stomacher bag containing 225ml of peptone water and homogenized for 3 minute using a stomacher. From the original homogenate, a loopful aliquot was spread on mannitol salt agar (MSA) and incubated from 24-48 hours at 37°C. Due to high salt concentration of MSA, it may not support the growth of weak and injured bacteria during sample processing and may result to false negative growth. In order to avoid this problem, 50ml of the original homogenate was directly incubated for 24-48 hours at 37°C and a loopful of inoculum from enrichment broth was also cultured on MSA. Pure cultures of presumptive colonies (yellow colonies on MSA) were streaked on nutrient agar and incubated for 24-36h at 37°C for Gram stain and further biochemical tests (catalase, coagulase test and oxidation–fermentation test). In addition, presumptive colonies was also inoculated to blood agar plates (5% difibrinated sheep blood) and plates were incubated aerobically at 37°C and examined after 24 hours of incubation for growth and hemolytic pattern of S. aureus.
Enumeration of Staphylococcus aureus count
In addition to identification, microbial counts of S. aureus were conducted on MSA by using spread plate count method. After confirming the sample was positive for S. aureus, tenfold of serial dilutions from the original homogenate were prepared and a 0.1ml sample from serial dilutions was spread on MSA and incubated from 24-48 hours at 37°C. Presumptive colonies were undergoing confirmatory test and golden yellow colonies on MSA with catalase and coagulase positive isolates, and complete hemolysis on blood agar were identified as S. aureus ount and the number of cfu/g of the test sample was calculated by the formula as described below [22, 23].
cfu per gram of sample = c/d *v
Where:- c = is the number of colonies on the counted plate,
d = the dilution rate of the counted plate and
v = the inoculated volume of this dilution
Anti-microbial susceptibility testing
All positive isolates of S. aureus were subjected to antibiotic susceptibility test by using the Kirby Bauer disc diffusion method as per Clinical Laboratory Standards Institute (CLSI) of USA guidelines on Mueller-Hinton agar (MHA). The antibiotics were selected based on the groupings of antibiotic agents with United States Food and Drug Administration clinical indications that should be considered for routine testing and reporting on non-fastidious organisms. One representative antibiotic agent from each subclass of antibiotics groups, commonly used and most available antibiotics for treatment of staphylococcal related diseases in animal and human was selected. Based on the above criteria, 9 antibiotics [chloramphenicol (30µg), ciprofloxacin (5µg), cefoxitin (30µg), clindamycin (2µg), erythromycin (15µg), gentamycin (10µg), penicillin (10 units), tetracycline (30µg) and trimethoprim-sulfamethoxazole (1.25/23.75µg)] were selected for this study [24].
For susceptibility test, three to five well-isolated colonies of the same morphological type were selected from nutrient agar plate culture and transferred into test tubes containing sterile saline and mixed thoroughly. The density of the suspension was adjusted to McFarland 0.5 by addition of saline or more S. aureus colony. A sterile swab was dipped into the suspension and the excess of inoculums were removed by pressing it against the sides of the tube to avoid over-inoculation of plates. The inoculums were spread evenly over the entire surface of the agar plate by swabbing in three directions. Antibiotic discs were applied firmly on the agar surface and incubated for 24h at 37oC. The diameter of the zone of inhibition around the disc was measured using ruler in millimeter (mm) and interpreted according to the standard of CLSI as susceptible, intermediate or resistant [24-26]. Those isolates showing resistance to three or more antibiotics were considered as multiple drug resistant (MDR) [20].
Data quality assurance
The data quality and the reliability of the study findings were assured by following standard operating procedures and the routine use of control bacterial strains. The sterility of prepared media was checked by incubating some randomly selected plates for 24-48 hours at 37oC. Uninoculated media was incubated as negative control to check for sterility. The quality of the culture media and test procedures were thoroughly checked using standard American Type Culture Collection (ATCC) strain of S. aureus (ATCC25923) as a positive control for screening tests, confirmatory tests and disk diffusion antibiotic susceptibility tests. Escherichia coli ATCC-25922 was used as a negative control for culture on mannitol salt agar.
Data management and statistical analysis
Raw data and laboratory findings were encoded into Microsoft Excel, exported into STATA software version 12.0 and analyzed using descriptive statistics such as frequency, percentages, mean and standard deviation (SD). In all the analyses, confidence level was held at 95% and p-value was assumed less than 5% (P<0.05).