Evaluation of Bacterial Load and Antibiotic Resistance Pattern of Staphylococcus aureus from Ready-to-Eat Raw Beef in Bahir Dar City

Staphylococcus aureus is one of the most important causes of foodborne intoxication and most frequent antibiotic resistant pathogen in the world. Regular evaluation of the current safety status of food is a pro-active measure to minimize the possible danger of foodborne pathogens. Therefore, this study was conducted on to assess the bacterial load and antibiotic resistance profile of S. aureus from ready-to-eat raw beef in Bahir Dar city. This cross-sectional study was conducted from October 2018 to April 2019 by collecting a total 101 raw beef samples from butcher shops using simple random sampling method. Isolation and microbial load determination of S. aureus using was performed conventional culture method as well as antibiotic susceptibility test was conducted by using Kirby Bauer disk diffusion method on Mueller-Hinton agar. The data were analyzed by using STATA software version 12.0.

methods should be employed to prevent S. aureus intoxications in foods.

Background
Animal origin food items are rich sources of nutrients and provide a variety of micronutrients to humans that are not obtained in plant-derived foods [1]. Meat is one of the most nutritive and favorite animal-source foods. Due to its high water content (0.99 water activity), rich in proteins, minerals and other nutrients which are suitable for microbial growth, meat is highly perishable food that can cause infection in humans and also can lead to economic loss due to spoilage [2,3].
A large number of foodborne zoonotic diseases often occur due to the consumption of contaminated animal origin foods such as meat and milk [4]. Among the bacteria predominantly involved in foodborne diseases (FBD), S. aureus is one of the leading causes of foodborne intoxication throughout the world resulting from the consumption of preformed staphylococcal enterotoxins [5][6][7]. Due to poor hygienic practices and low level of awareness, this problem is worse in developing countries.
Staphylococcal foodborne intoxication (SFI) is often associated with the ingestion of highly heat stable staphylococcal enterotoxins. The acceptable level of S. aureus in ready-to-eat food should be below10 3 colony forming unit per gram (cfu/g) of food. If the amount of bacteria is greater than 10 4 cfu/g, the food is unsatisfactory and potentially hazardous for health and/or unfit for human consumption [8]. Ingestion of nanogram to a microgram of staphylococcal enterotoxin contaminated food can cause serious illness ranging from minor skin infection to life threatening diseases [9].
Antimicrobial resistance is a serious threat to public health across the globe. A wide variety of antimicrobial drugs are employed to treat S. aureus infections. However, emergence and spread of antimicrobial resistant S. aureus isolates constitute a global challenge for the effective treatment and control of these infections [10][11][12].
In Ethiopia, the burden and public health impact of foodborne illness related with S. aureus infection is poorly understood. However, the epidemiology of this bacteria and the widespread habit of raw meat and milk consumption in the population are suggestive of the risk of acquiring S. aureus [ 13]. Consuming raw beef in the form of simple cut strips of meat which is locally called "Kurt", is a common habit and is an indication of wealth in Ethiopia [14]. Moreover, raw beef is available in open-air local butchers of Ethiopia without the cold-chain process which could be serving as a potential source for foodborne illnesses [15,16].
Besides the prevalence of S. aureus in meat reaches up to 40% [17] in butcher shops of Mekelle city, there is a paucity of data regarding to S. aureus from ready-to-eat raw beef in Bahir Dar city. Therefore, this study was focused on isolation and identification of S. aureus, measuring its magnitude and antibiotic resistance pattern from ready-to-eat raw beef to provide useful information regarding to staphylococcal loads and their antibiotic resistance profile in Bahir Dar city.

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°N orth 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.
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  [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 37 o C. 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).

Isolation rate of Staphylococcus aureus
In the present investigation, from a total of 101 ready-to-eat raw beef samples subjected for cultural and biochemical isolation, 55(54.45%) were positive for S. aureus which suggests that the bacteria could be a major food contaminant in butcher's shops of Bahir Dar city.

Microbial load of Staphylococcus aureus
The enumeration of S. aureus was performed on MSA using spread plate technique. The minimum and maximum S. aureus counts in ready-to-eat raw beef samples of Bahir Dar city were 2.48 and 5.08 (log 10 cfu/g) respectively with a mean and standard deviation of 3.40 ± 0.63 (log 10 cfu/g). Based on CFS (2014) microbiological guidelines for food, none of the butcher shops had good microbial level (below 100cfu/g) and majority of them (49%) had unsatisfactory level followed by acceptable microbial level (38%) for S. aureus counts ( Figure 1). The mean count of S. aureus in this study was in line with 3.88log10cfu/g reported from fresh meat Bahir Dar City [27]. About 13% of butcher shops had unacceptable and potentially dangerous bacterial load. If the bacterial count exceeds the above standard in fresh meat, then the meat is not acceptable and this indicates alarm signals on meat hygiene along meat chain from abattoir to butcher shops [28].
The mean count of S. aureus this study was lower than the findings (5.61±0.10 log10 cfu/cm 2 for cutting boards and 6.43±0.34 log10 cfu/cm 2 for butchers' hands) from retail houses of Jigjiga town [29]. These variations of bacterial load might be due the difference of meat processing, handling practices and sanitary standard operating procedures along the meat production chain.
In most of the retail points, meats were seen on the ground and left to the mercies of the environment which can create an avenue for microbial pathogens to proliferate on it.
These high bacterial loads could affect the average shelf life of the meat and increase the chances of spoilage.

Antibiotic susceptibility profile of Staphylococcus aureus
All the 55 positive samples of S. aureus isolates were undergo in-vitro antibiotic susceptibility test on MHA medium by using disc diffusion technique. The isolates were completely susceptible to ciprofloxacin followed by gentamycin (89.09%) ( Table 1). This finding was agreed with the finding that all S. aureus isolates from meat sample in Addis Ababa were susceptible (100%) to ciprofloxacin [30]. This finding was also compatible with the previous research output from dairy farm and abattoir in Addis Ababa that 97.7% of the isolates were susceptible to ciprofloxacin [20]. The reason for ciprofloxacin susceptibility of the isolates could be due to the fact that ciprofloxacin is relatively expensive and newly introduced antibiotic as compared to the other common antibiotics [27].
Majority of the bacterial isolates (92.73%) were found to be resistant to the antibiotic penicillin and 74.5% of the isolates were resistant for cefoxitin. The present study was in harmony with report indicated that 95.3% of the isolates were resistant to penicillin [20].
The reason for high resistance of penicillin and other β-lactam antibiotics could be that they are the most commonly used antibiotics for the treatment of infection in humans and animals in Ethiopia [20]. The present study revealed that about 98% of S. aureus isolates had high resistance to two or more drugs due to the fact that there is frequent irrational antimicrobial use and misuse behavior in the country [20] (Figure 2).

Multidrug resistance profile of S. aureus
From the tested 55 isolates of S. aureus, 46 (83.64%) isolates showed MDR. Nineteen isolates were resistant to three antibiotics, similarly twenty and seven isolates were resistant to four and five antibiotics respectively ( Table 2). The presence of MDR of S.
aureus isolates in this study area indicates the possible significant risk of the resistant strain along the studied beef line.

Ethical approval and consent
The study was conducted after the protocol was ethically reviewed and approved by Butchery's owner permission was asked to take raw beef sample with full consent by explaining the objectives of the study, their right and their participation is fully voluntarily. To ensure confidentiality, any personal or butchery identifying information was not collected and it was managed with unique code.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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

Funding
The fund for this study was obtained from Mizan Agricultural Technical Vocational and

Educational Training College and Bahir Dar University
Authors' contributions BT developed research idea and conducted the main study. TA and BA supervised, commented and corrected the research protocol, data analysis and all the write-up processes. SY had major contribution during data collection and laboratory investigation.
All authors read and approved the final manuscript. Figure 1 Microbial quality levels of butcher shops based on S. aureus count from raw beef in Bahir Dar city Drug-resistance patterns of S. aureus isolates from raw beef sample in Bahir Dar city