Microbiological quality and safety of Ready- to- eat foods from restaurants and food establishments in Yirgalem town Southern, Ethiopia

Background: Foodborne illnesses are considered as one of the most important public health problems particularly in developing countries like Ethiopia. This study aimed to determine the microbiological quality and safety of ready-to-eat foods in Yirgalem town, southern Ethiopia from November 2016 to August 2017. Methods: The collection of ready-to-eat food samples and laboratory-based microbiological analysis was used as the study design. A total of 160 food samples comprising of 40 ‘Injera firfir’, 40‘Bayeaynet’, 40 Vegetables and 40 Spaghetti were collected and analyzed for microbial contamination following standard microbiological methods. Ten grams of each food sample was transferred into 90 ml of buffered peptone water and homogenized for 5 minutes using a vortex mixer. The homogenates were serial diluted up to 10-7 and a volume of 0.1ml aliquot was spread plated on pre-solidified media of Aerobic plate count agar, MacConkey agar, Mannitol salt agar, and Salmonella-Shigella agar and incubate at 35-37oc for 24 hrs. Also, Potato Dextrose Agar was used for the isolation of fungi. Data were entered into Microsoft Excel and analyzed using SPSS version 20.0. Results: All the collected food samples were subjected to total aerobic mesophilic bacteria, Coliform bacteria, Enterobacteriaceae, Staphylococcal, Yeasts, and Molds counts. Accordingly, the mean counts expressed as log10 CFU/g of food for each group of the organism were 7.90 ± 0.71, 4.31±1.30, 4.32 ± 1.30, 6.70 ± 0.34 and 4.5 ± 1.01, respectively. The highest bacterial load 162(28.9%) was detected in ‘Injera firfir’ whereas the lowest 108(19.2%) case was investigated in Spaghettis. Regarding the food safety issue, the frequency of S. aureus, E. coli and Salmonella spp in the food samples were 54.4%, 43.8%, and 0.6%, respectively. Conclusion: The high microbial load and existence of foodborne pathogens in ready-to-eat foods in Yirgalem town, Southern Ethiopia is calling for the creation of awareness among restaurant and food establishment owners and food handlers concerning the hygienic practice.

4.31±1.30, 4.32 ± 1.30, 6.70 ± 0.34 and 4.5 ± 1.01, respectively. The highest bacterial load 162(28.9%) was detected in 'Injera firfir' whereas the lowest 108(19.2%) case was investigated in Spaghettis. Regarding the food safety issue, the frequency of S. aureus, E. coli and Salmonella spp in the food samples were 54.4%, 43.8%, and 0.6%, respectively. Conclusion: The high microbial load and existence of foodborne pathogens in ready-to-eat foods in Yirgalem town, Southern Ethiopia is calling for the creation of awareness among restaurant and food establishment owners and food handlers concerning the hygienic practice. Keyword: Microbial quality, Yirgalem town, Southern Ethiopia Background The fight against foodborne diseases is facing new challenges due to the globalization of the food market, climate change and changing patterns of human consumption [1]. The foodborne disease remains a major source of morbidity and mortality in the general population, mainly in susceptible 3 groups, such as infants, elderly and the immunocompromised people [2]. According to the World Health Organization (WHO), 2007 report, up to 1.5 billion cases of diarrhea and more than 3 million deaths that occur in children every year are as a result of food and water contamination [3]. In the United States of America (USA) it is estimated that foodborne diseases result in 76 million illnesses, 325,000 hospitalizations and 5,000 deaths each year [3,4].
Although foodborne illnesses cause substantial morbidity in developed countries, the main burden is borne by developing nations. In Southeast Asia, approximately 1million children below the age of five years die each year from diarrheal diseases due to contaminated food and water [5]. Several devastating foodborne outbreaks have been reported from the African continent; for example, in 2004, Kenya experienced an acute aflatoxicosis outbreak which was attributed to maize whereas, in 2007, Angola registered 400 cases of bromide poisoning, associated with the use of sodium bromide as cooking salt [6].
Foodborne illness is also one of the common problems in Ethiopia because of the prevailing poor food handling and sanitation practices, inadequate food safety laws, weak regulatory systems, lack of financial resources to invest in safer types of equipment, and lack of education for food-handlers [7]. National Hygiene and Sanitation Strategy program [8] reported that about 60% of the disease burden was related to poor hygiene and sanitation in Ethiopia. Studies conducted in different parts of the country have shown that the poor sanitary conditions of catering establishments and the presence of pathogenic organisms like Campylobacter, Salmonella, Staphylococcus aureus, Bacillus cereus and Escherichia coli, [9,10,11,12,13] make foodborne illness more worsen. More aggravated situations and challenges prevailing in the country where food safety issues are not well understood and have received little attention. Therefore, this study aimed to assess the microbiological quality and safety of ready-to-eat foods from restaurants and food establishments in Yirgalem town, Southern Ethiopia.

Study area and period
The study was conducted at Yirgalem town, located 275 km south of Addis Ababa; the capital city of 4 Ethiopia. The town's geographical coordinates are 7 o 41'N latitude, 36 o 50'E longitude, and an average altitude of 1, 780 m above sea level. It lies in the climatic zone locally known as "Woyna Daga" (1,500-2,400 m above sea level) which is considered ideal for agriculture as well as human settlement. The town is generally characterized by a warm climate with a mean annual maximum temperature of 30°C and a mean annual minimum temperature of 14°C. The annual rainfall ranges from 1138-1690 mm. The maximum precipitation occurs during the three months from June through August, with minimum rainfall occurring in December and January. The study was conducted between November 2016 to August 2017.

Sample collection
A total of 160 food samples comprising of 40 Bayeaynet, 40 Injera firfir, 40 Vegetables, and 40 Spaghetti, were collected from different restaurants and food establishments in Yirgalem town, southern Ethiopia between November 2016 and August 2017. Food samples were collected using food serving utensils and placed into a sterile aluminum plate at a time between 5 PM and 6 PM after a brief description of the study purpose for the restaurant and food establishment owners. The samples collected were transported to the Yirgalem Hospital Medical College, Microbiology Laboratory in the date of collection using icebox and were kept in the refrigerator at 4 o C until microbial analysis was conducted. The microbial analysis was conducted within one to three hours of collection.

Sample processing
Twenty-five grams of each food sample was cut into small pieces using food cutter and was added into 225 ml of 0.1% sterile peptone buffer water and homogenized for 2 minutes using a stomacher.
One ml of the resultant homogenate was added into 9 ml of sterile 0.85% saline in a test tube and serially diluted up to 10 -7 .

Microbial analysis
Approximately 0.1 ml aliquot portions of the dilutions were spread onto duplicate sterile plates of plate count Agar, MacConkey Agar, Salmonella-Shigella Agar and Mannitol salt Agar, for total aerobic plate count, Coliform count, Enterobacteriaceae count, isolation of Salmonella and Shigella and isolation of Staphylococcus aureus, respectively. Also, Potato Dextrose Agar was used for the isolation of fungi. All cultures were incubated at 37 o c for 24-48 hrs except that of Potato Dextrose Agar which was incubated for 3 to 5 days at 25 o c. All the culture plates were checked for microbial growth when the appropriate time of the incubation was achieved. Thereafter, colonies between 30-300 were counted using colony counter (Gallenkamp, England) and the counts were expressed as colonyforming units per gram (CFU/g) of food. Cultures were purified by repeated plating and maintained on appropriate slants at 4 0 C (14). Finally, the pure isolates were identified through gram staining and biochemically using (Catalase test, Oxidase test, coagulase test, Triple sugar iron agar, Lysine iron agar, Urea agar, Simmons Citrate agar, and SIM media) following standard microbiological methods (15,25). Moreover, fungal isolates were identified based on their macroscopic and microscopic features. Reference was made standard identification keys and atlas.

Statistical Analysis
The data obtained were analyzed using SPSS software version 20.0. The significance of differences was considered at a 95% confidence interval (P < 0.05).

Ethical Consideration
The study was approved by the Institutional Review Board of Yirgalem Hospital Medical College and an official permission letter was obtained from the Yirgalem town Hotel and tourism office. Written informed consent was also obtained from restaurant and food establishment owners after a brief 6 explanation of the objectives and benefits of the study.

Data quality control
Aseptic techniques were employed in every step of the study. Samples were transported in an icebox and were processed without delay and in replicates. To ensure sterility, materials and media were autoclaved at 121 o c for 15 to 20 minutes. The media were checked for sterility by incubating 5% of the batch at 37 o c for 18-24 hrs.

Operational definitions
Ready-to-eat foods: Foods that are available directly for consumption without further processing  Table 2). The highest isolates were detected in Injera firfir followed by Bayeaynet.

Discussion
Foodborne illnesses caused by Salmonella spp, S. aureus, and E. coli represent a major public health problem worldwide. These pathogens are transmitted mainly through the consumption of contaminated food and the presence of these organisms in ready to eat foods has relevant public health implications [16]. In the present study, it was found out that almost all the food samples collected from restaurants and food establishments in Yirgalem town southern Ethiopia had harbored various pathogenic and indicator microorganisms. This finding was in agreement with the study reported from Gondar, Ethiopia where over 82.8% of the food samples analyzed for microbial contamination had contained various pathogenic microorganisms [17].
In this study, a total of 560 AMB were isolated from the collected food samples. The isolates were included Staphylococcus spp 279(49.82%), EB 125(22.32%), Coliform70 (12.50%), Micrococcus spp 32(5.71%), Pseudomonas spp 27(4.82%) and Acinetobacter spp 27(4.82%). The presence of coliform in ready to eat foods can be linked to contamination resulted from inappropriate processing, incomplete heating, use of contaminated water during preparation and washing or secondary contamination via contact with contaminated types of equipment such as chopping boards, knives, and serving wares [17].
In this study, Escherichia coli were detected in 43.8% of the food samples. This detection rate was consistent with the previous study carried out in Amravati city and Gondar, Ethiopia where 41.0% and 46.3% of the food samples analyzed were found contaminated with E. coli, respectively [17,18]. The presence of E. coli in ready to eat foods might attribute to the heat processing failure or postprocessing contamination, fecal contamination and poor hygienic practice of food handlers [19].
In the current study, the prevalence of S. aureus in the whole food samples was 54.4%. This result was greater than the study result of [22] where 56.2% of 'Bonbolino' samples were contaminated with 8 S. aureus. However, S. aureus frequency in this study was consistent with the findings of [18] from Gondar, Ethiopia where 53.7% of the street vended food samples were contaminated with S. aureus.
The presence of S. aureus is an indication of contamination from the skin, mouth or nose of food handlers through coughing and sneezing during food handling and processing [22].
In this study Salmonella, spp was detected in 0.6% of the food samples. This is in agreement with previous work done on 'Sambusa' and 'Macaroni' in Ethiopia [23]. The presence of Salmonella spp in foods ready to eat might be an indication of the existence of fecal contamination.
In the present study, 'Injera firfir' and 'Bayeaynet' were highly contaminated food items with bacteria and fungi. This could be due to the method of handling and preparation of the foods. 'Injera firfir' and 'Bayeaynet' are more frequently handled by bare hands when compared with other food groups.
Hence, food handlers may be the source of food contamination either as carriers of a pathogen or through poor hygienic practices. All food handlers have a basic responsibility to maintain a high degree of personal cleanliness and observe hygienic and safe food handling practices [19,24].

Limitations
Even though the present study tried to address an important issue concerning food contamination, it has some limitations. Antimicrobial sensitivity for the isolates was not done because of the inaccessibility of the appropriate disc during the study period. Due to a shortage of enrichment and selective media for isolation, we did not other foodborne pathogens.

Conclusions
The results of this study demonstrated that the ready to eat foods in Yirgalem town southern Ethiopia were contaminated with different pathogenic bacteria. The existence of pathogenic bacteria such as S. aureus, E. coli, and Salmonella spp in foods could induce potential health problems for consumers.   Where: EB, Enterobacteriaceae; Numbers in the parenthesis are a percentage of the total isolates of respective species