Public health risks and bacterial safety of fruit juices prepared in Axum town, north Ethiopia

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1 Research Article Public health risks and bacterial safety of fruit juices prepared in Axum town, north Ethiopia Haftom Kebede 1 *, Haftom Hadush 2, Teklay Gebrecherkos 3, K. Krishna Chaithanya 4 ABSTRACT Objective: Improperly prepared fresh fruit juices are recognized as one of the major causes of food-borne illnesses. Therefore, this study was aimed at evaluating the public health risks and microbial safety of fruit juices prepared in Axum town, North Ethiopia and their hygienic conditions of preparations. Materials and Methods: Eighty fruit juices samples were collected from 20 cafés and juice houses of Aksum town and analyzed for fecal coliform count (FCC), using most probable number (MPN) method and the total colony count of Staphylococcus aureus, Salmonella spp., Shigella spp., and Escherichia coli was done by spread plate method using plate count agar for bacteria, and further the bacterial isolates were tested for their sensitivity to common antibiotics using the disc diffusion method on Mueller-Hinton Agar. Results: Results shown that the mean FCC of mango, Avocado, papaya, and Guava were 68, 80, 58, and 65 cfu/ml, respectively. The bacterial isolates were identified as S. aureus, E. coli, Shigella spp., and Salmonella spp. All the juice samples tested were contained all the four isolated bacteria, except Avocado and mango which were negative for Salmonella. These bacterial isolates were susceptible to ampicillin, gentamicin, ciprofloxacin, and chloramphenicol, and resistance to erythromycin, and resistance to ciprofloxacin, amox-clavul acid, ceftriaxone, and tetracycline. The results also showed that the microbial loads of most of the fruit juices were higher than the specifications set for fruit juices sold in the Gulf region and other parts of the world. Conclusion: Most venders obtained fruit from the open market, and all juice makers lacked special training in food hygiene and safety. Therefore, regular training and health education on food hygiene and safety are recommended for juice handlers to improve the quality of fresh fruit juices in the study area. KEY WORDS: Antibiotic, Antibiotic susceptibility, Fruit juices, Hygiene, Microbial safety INTRODUCTION Fruit juices are nutritious which offer great taste and health benefits. [1] These fruit juices are processed under hygienic condition, they are well recognized for their nutritive value, mineral vitamins and secondary metabolites such as flavonoids and tannins played an important role in enhancing consumers health through inhibition of breast cancer, congestive heart failure, and urinary tract infection. Fruit juices are common beverages in many countries of the world, and are sold at all public places, in restaurants and cafeterias. There are several reports of food-borne illness associated with the consumption of contaminated fruit juices at Access this article online Website: jprsolutions.info ISSN: several places among the world. [2,3] This means that there is a chance that the product may contain bacteria which could be harmful to our health. [4] In addition, poor handling and processing of fresh fruit juices are some of the main cause of food associated illness to the community who live in developing countries such as India and Ethiopia. In most cases, a number of pathogenic organisms are isolated and identified from locally prepared fruit juices, due to unhygienic fruit handling in the unsanitary environmental conditions under which the vendors operate the juices become contaminated with harmful bacteria. [5] Such juices have shown to be potential sources of bacterial pathogens notably Escherichia coli 0157:H7, species of Salmonella, Shigella, and Staphylococcus aureus. [6] Today, the incidence of antibiotic-resistant bacteria which have been associated with foodstuff is a worldwide phenomenon and it is becoming a major 1 Department of Biology, College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia, 2 Department of Biomedical Sciences, College of Health Sciences, Aksum University, Axum, Ethiopia, 3 Department of Biomedical Microbiology, School of Biomedical Laboratory Sciences, University of Gondar, Ethiopia, 4 Department of Chemistry, College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia *Corresponding author: Haftom Kebede,Department of Biology, College of Natural and Computational Sciences, Aksum University, Axum, Tigray Region, Ethiopia. Phone: tom.kebede@ gmail.com Received on: ; Revised on: ; Accepted on: Journal of Pharmacy Research Vol 12 Issue

2 public health threat, [7] as these organisms have been isolated from wide range foodstuffs consumed by man. The relevance of information obtained on the resistance of bacteria to antibiotics is to appreciate the magnitude of the problem and establish baselines for taking action. [8] There are different types of tropical fruits (e.g., orange, grape, pineapple, banana, guava, and watermelon) readily available for the production of fruit juices. The juice may be produced from single fruit or combination of fruits and sold by street vendors. [9] Although it is difficult to prove a direct role of drug resistance in bacteria contaminating food items with increased clinical cases of resistant infections, the presence of such bacteria in food items could play a role in the spread of antimicrobial resistance among food-borne pathogens. [10] In Axum Town (Tigray region, Ethiopia) there is a great demand for the fruit juices, most restaurants and juices houses, are in apparently unhygienic conditions, due to mishandling of juices. However, in recent years the increasing consumer awareness has emphasized the need for microbiologically safe food. However, the magnitude of the problem in Ethiopia particularly in Axum Town is not yet determined. However, the present study was aimed to assess the type of microorganisms that can be found in commonly used fruit juices from juice houses, restaurants, and cafés setting and their drug sensitivity pattern. This was also aimed to minimize the microbial risk of fruit juice associated transmission of pathogens in the settings where many people have common services. It may also further help for those decision makers to develop guidelines how to locally prepared fruit juice and other food associated infections can be prevented. MATERIALS AND METHODS Description of the Study Area A cross-sectional study was conducted from February 1, 2017, to May 30, 2017, at Axum Town, North Ethiopia. Axum Town is 1040 km far from Addis Ababa and is located at Latitude 14, 1297 ( N) and Longitude 38,7158( E) and is the administrative center of Central Zone of Tigray. It has 4 kebeles and approximately 80, ,000 populations. There are many juice houses, restaurants, and cafeteria that prepare fruit juices that can be consumed by visitors and people of Axum town. Restaurants and cafeteria use tap water for preparation of juice, half of them use antiseptic for cleaning facilities and they located near roadside where environmental microflora harboring over the fruits and utensils surfaces in the form of dust exploded by the traffic heavily contaminated the fruit juices. Mango and avocado fruit juices are the most popularly consumed by juice types in the town. Study Design The design of the study was cross-sectional survey involving questionnaire administration to determine the factors related to microbiological quality and safety of fruit juice, and laboratory investigation to determine the microbial load, pathogenic microbes and antibiotic susceptibility of the isolated bacterial species from fruit juice samples in Akum town. Sample Size A total of 80 fruit juice samples of four types (avocado, mango, papaya, and Guava) from 20 sites, i.e., 4 samples of each juice type, were collected. This was achieved by collecting one sample for each type of fruit juice. Data Collection Two basic data collection methods were used in this study: Questionnaire administration and laboratory experiments. Questionnaire Administration A structured questionnaire was distributed to all (20) juice makers, who prepare juice in 20 café, juice houses, and restaurants. The questionnaire was used to obtain information on the demographic characteristics of the respondents, sources of fruit, storage conditions, water source for juice preparation, as well as for cleaning purpose, cleaning habit of the juice makers, the practice of washing the fruits before making juices, the practice of cleaning the juice processing equipment, whether or not the juice makers have had training in food hygiene and safety, awareness about microbial contamination and its consequences. Laboratory-based Experiment The laboratory-based experiment involved avocado, papaya, Guava and mango juice sample collection, sample processing, isolation, and identification of microorganisms from the juice samples; and testing pathogenic bacteria for their antibiotic sensitivity. Sample Collection Eighty samples of avocado, papaya, Guava, and mango of locally prepared unpasteurized fruit juices were collected from Aksum town in four rounds, i.e., four juice samples (one avocado, one papaya, one Guava, and one mango juice) from five cafes or restaurant in different days. All the samples were collected on a voluntary basis from participating restaurants and cafes in sterile plastic container (250 ml), aseptically labeled, and immediately transported to Aider Referral Hospital, College of Health Sciences, Department of Medical Microbiology, Mekelle University Laboratory, in an icebox where they were processed immediately. 510 Journal of Pharmacy Research Vol 12 Issue

3 Sample Processing For analysis, 25 ml of fruit juice was measured using measuring cylinder and transferred to 225 ml of sterile peptone water and homogenized by shaking in an aseptic environment, which was achieved by cleaning and disinfecting using alcohol as well as using Bunsen burner flame. Serial dilutions (10 1, 10 2, 10 3, and 10 4 ) were prepared by taking 1 ml from a homogenized sample and adding to sterile test tube containing 9ml of sterile peptone water and mixing properly. [11] Bacterial Analysis of Fruit Juice Microbiological analysis was done using appropriate media designed for enumeration and identification of different microbial groups following standard procedures. [12] The total colony count was done by spread plate method using plate count agar for bacteria. [13] Fecal coliform counts (FCC) were determined using the most probable number (MPN) method. [14] All inoculated media were incubated at appropriate temperatures for the required period of time. After enumeration, colonies were randomly picked from countable plates and further purified by repeated plating on plate count agar. The resulting bacterial isolates were then identified following standard microbiological procedures as described by Cheesbrough. [15] Enumeration of Bacterial Isolates Staphylococci count Enumeration of Staphylococci was done using Mannitol Salt Agar (MSA) in four replicates following standard methods and procedures. From appropriate dilutions 0.1 ml of sample fruit juices were spread plated on MSA and then incubated at 30 C for h. Then, each plate was observed after h of growth and presumptive colonies were counted [16] for confirmation of S. aureus, coagulase test was performed. To do this, inoculum from each presumptive colony of MSA plate was transferred to a separate tube of Brain Heart Infusion (BHI) broth and incubated at 35 C for h under aerobic condition. Then, 0.2 ml of BHI broth culture was transferred into sterile mm tubes containing 0.5 ml certified coagulase plasma and mixed thoroughly. The mixture was incubated at 35 C and examined after 1 h and 4 h. A firm clot, which did not move when the tube was tipped on its side (coagulase reaction), was considered a positive test for S. aureus. If no clot was observed, it was considered as a negative test for S. aureus. Gram staining was also done for confirmation of S. aureus by preparing smears from the deep yellow opaque colonies. Total Coliform Count The three-tube procedure using lactose broth [17] in three replicates was used to detect the coliform and determine the MPN of coliform. The MPN method was used as a combination of presumptive, confirmed, and complete incubated for 48 ± 3 h at 35 ± 0.5 C and examined for gas formation. Formation of gas in any amount in the inverted vial at any time within 48 ± 3 h was recorded as a positive confirmed test. Negative test tubes were re-incubated for additional 24 h. Number of positive tubes for each dilution was recorded. Tubes showing positive results were streak plated on eosin tests. 1 ml of each of the 10 3, 10 4, and 10 5 dilutions was inoculated into three test tubes of LB each containing Durham s tube. After incubating for 24 h, the number of tubes in each set of three that showed positive for acid and gas production was recorded. Negative test tubes were re-incubated for additional 24 h. Each presumptive positive tube of lactose broth was gently swirled and a loopful of each positive culture was transferred to tubes of brilliant green lactose bile 2% broth (BGLBB) using a sterile inoculating loop. Inoculated BGLBB tubes were eosin methylene blue (EMB) agar and incubated at 37 C for 48 h. From each EMB agar plate a typical coliform colony (pink to dark red with a green metallic surface sheen) was transferred to a tube containing lactose broth and a nutrient agar slant, incubated at 35 ± 0.5 C for 48 ± 3 h. Calculation of MPN was done from the completed test results using the formula employed by Thomas. [14] MPN = P TN Where: P = the number of positive tubes T = Total quantity of sample in all tubes in ml N = Total quantity of sample in negative tubes in ml. FCC Fecal coliforms were obtained by MPN technique. 1 ml of each of the 10 3, 10 4, and 10 5 dilutions was inoculated into three test tubes of LB with Durham s tube and incubated at 37 C for 48 h. [18] Each presumptive positive tube of lactose broth was gently swirled and a loopful of each positive culture was transferred to tubes of EC broth using a sterile inoculating loop. Inoculated EC broth tubes were incubated for 48 ± 3 h at 45 ± 0.5 C in water bath. Gas production in an EC broth culture was considered as a positive fecal coliform reaction. Only tubes, which were positive in the EC medium within 24 h, were used in the calculation of fecal coliforms. The presence of fecal coliforms was confirmed by streaking from positive EC broth culture on EMB agar plates. Bacterial colonies developed were considered as fecal coliforms. [10] Biochemical Test Catalase, oxidation fermentation, carbohydrate utilization, indole production, citrate utilization, growth on MacConkey agar and methyl red- Voges Proskauer tests were carried out according to standard procedure described in Roberts and Greenwood. [11] Journal of Pharmacy Research Vol 12 Issue

4 Detection of Bacterial Pathogens Pathogenic bacteria such as Salmonella, Shigella, E. coli, and S. aureus were detected according to the procedures outlined by Food and Drug Administration. [19] For detecting, the presence of Salmonella and Shigella 25 ml of juice sample was added into 225 ml of sterile peptone water and homogenized by shaking. The resulting dilution was then serially diluted up to 10 5 dilution. From the 10 1 and 10 2 dilutions, 1 ml was taken and inoculated into tubes of LB and incubated at 37 C for 48 h. A loopful of sample from each culture was then transferred to Rappaport Vassiliadis broth and incubated at 42 ± 0.2 C for 24 h in water bath. Positive samples were confirmed by streaking on Hektoen Enteric Agar and then biochemically tested for the presence of Salmonella and Shigella. In all cases, for confirmation of the pathogens, typical colonies were identified based on cultural microscopic and biochemical characteristics. [12] Antibacterial Susceptibility Test All isolates of pathogenic bacteria were tested for their sensitivity to antibiotics by means of the disc diffusion method on Mueller-Hinton Agar (Difco, Detroit, MI) as described previously by Bauer et al. [20] All disks used in the disk diffusion test were obtained from BECTON, USA, in the following concentrations: Ciprofloxacin (5 µg), ceftriaxone (30 µg), gentamicin (10 µg), cotrimoxazole (25 µg), erythromycin (15 µg), amoxclavul acid (30 µg), ampicillin (10 µg), chloramphenicol (30 µg), and tetracycline (25 µg). Briefly, five colonies of each isolate were introduced into 5 ml of nutrient broth, incubated for 4 h, and the culture turbidity was adjusted to a 0.5 McFarland standard. Sterile cotton swab was dipped into the suspension and spread evenly over the entire Mueller-Hinton Agar surface. The antibiotics impregnated discs were then placed onto the surface of the inoculated plates and incubated at 37 C for h. After incubation, diameters of the zones of inhibition were measured in mm and interpreted as susceptible, intermediate, and resistant. Data Analysis After all data were collected, each measurement of the different variables was systematically organized into tables and figures and subsequently subjected to statistical analysis. Data analysis was done using the SPSS computer software version ANOVA was used to compare mean values among sampled juices. P < 0.05 was considered statistically significant. RESULTS AND DISCUSSION Demographic Characteristics of Respondents Among the 20 juice makers more than half (70%) of the fruit juice makers who participated in this study were females and 12 (60%) of them were younger than 35 years of age. 25% had education higher than primary education; 50% had primary education while only 25% had non-formal education [Table 1]. A demographic characteristic of respondents was disagreeing with the work of Tsige et al.[1] (2008) who reported that all the 90 fruit juice makers interviewed were females and 87.5% had education, higher than primary education, 9.17% actually acquired primary education while only 3.33% had no formal education. Level of Awareness Toward Bacterial Contaminants, Food Safety, and the Hygienic Conditions of the Fruit Juice Processing As showed in Figure 1, The source of fruits used for the processing of juices was primarily from the open market (85%) while some juice makers (15%) got their fruits directly from producers who were their routine suppliers. The temporary storage sites of fruits were shelves (25%), baskets (45%), and refrigerators (20%). 100% of the juice makers did not have training in food hygiene and safety; moreover, 75% of fruit juice makers were used protective cover during the preparation of fruit juices, and 85% of the respondents used hair cover during working [Table 2]. Figure 1: Bacterial analysis of fruit juice and antimicrobial susceptibility patterns of isolated bacteria from avocado juice, mango juice, papaya juice, and guava juice, (a) sample collection, (b) sample processing, (c) bacterial analysis of fruit juice, (d) antimicrobial susceptibility Table 1: Demographic characteristics of respondents in Aksum Town Variables Frequency (%) Age Below 35 years 12 (60) 35 and above years 8 (40) Gender Female 14 (70) Male 6 (30) Education status of juice maker Non formal education 5 (25) Elementary 10 (50) High school and above 5 (25) 512 Journal of Pharmacy Research Vol 12 Issue

5 All of the venders were using tap water for dilution of fruit juices and washing fruits before making juices with water only. All juice producers lacked special training in food hygiene and safety as it is indicated in this study and some of them (30%) had the awareness on the consequences of consuming contaminated foods [Table 2]. All juice producers lacked special training in food hygiene and safety as it is stated in this study and some 30% had the awareness on the consequences of consuming contaminated foods. The percentage of respondents on most of the possible factors affecting the quality of juice was in line with the work of Olorunjuwon et al., [21] who reported that source of fruits used for the processing of juices was mostly from the open market (85%); the temporary storage sites of fruits were shelves (35%), baskets (45%) and refrigerators (20%), and 100% of the juice makers did not have training in food hygiene and safety; very few had the knowledge of symptoms because of eating contaminated foods. Bacterial Analysis of Fruit Juice Total fecal count From the total 80 samples of Avocado, mango, papaya, and Guava juices 58 juices were above 100 MPN. The data reveal that all fruit juices samples collected from all houses were contaminated with coliform whereas all juice samples collected from most houses were contaminated with fecal coliform [Table 3]. It is contended that contamination is mainly due to the poor quality of water used for dilution as well as prevailing unhygienic conditions related to improper washing of fruits, and utensils, inadequate storage of these at ambient temperatures in unhygienic places, maintenance of premises and personal hygiene of vendors. In Bangladesh, Shakir et al. [5] showed the presence of E. coli ranging from 43 to >2400/100 ml in different types of vended squeezed fruit juices in Dhaka city. In India, Bagde and Tumane [22] reported that E. coli was heavily contaminated the fruit juices. The maximum permitted level of fecal coliforms for any ready to eat fresh juice is 100 MPN/25 g. Frequency of Bacterial spp. in Avocado Juice, Mango Juice, Papaya, and Guava Juice The biochemical test was performed for identification and characterization of bacterial isolates from locally prepared unpasteurized fruit juices samples. As shown Table 4 Four bacterial genera were isolated from the fruit juices, and these were characterized as S. aureus, E. coli, Salmonella spp., and Shigella spp. Among these isolates, the dominant organism was S. aureus with 35.5% (16/45) while the lowest was E. coli and Salmonella spp. with 5% (2/40). The finding was in line with the study of Olorunjuwon et al., 2014 [21] who reported that Klebsiella spp., Enterobacter spp., Bacillus cereus, Serratia sp., S. aureus, Penicillium Table 2: Level of awareness toward bacterial contaminants, food safety, as well as the hygienic conditions of the fruit juice processing Variable Frequency (%) Source of fruits Directly from producers 3 (15) Open market 17 (85) Temporary storage sites of fruits Shelf 7 (35) Basket 9 (45) Refrigerator 4 (20) Covering of hair during working Yes 17 (85) No 3 (15) Use of protective cloth during work Yes 15 (75) No 5 (25) Washing of equipment Water only 3 (15) Water and soap 10 (50) Water, soap, and bleaching agents 7 (35) Accessibility of fruits to flies Yes 7 (35) No 13 (65) Washing of fruits Yes 20 (100) No 0 (0) Training in food hygiene and safety Yes 0 (0) No 20 (100) Water source for juice preparation Tap water 20 (100) Well water 0 (0) Spring water 0 (0) Awareness that microorganisms can contaminate food Yes 6 (30) No 14 (70) Table 3: Fecal coliforms of avocado juice, mango juice, papaya, and Guava juices (MPN/25 g) Sample type Minimum FCC Maximum Number of sample with>100 MPN/25 g FCC Avocado Mango Papaya Zethun FCC: Fecal coliform count, MPN: Most probable number, +: Above spp., and Aspergillus niger were isolated from avocado juice. Adesetan et al., 2013, [23] also reported that S. aureus, Micrococcus sp., Bacillus subtilis, Lactobacillusspp., Streptococcus spp., E. coli, B. cereus, Klebsiella pneumoniae, Serratia plymuthica, Serratia ficaria, Proteus mirabilis, and Enterococcus faecalis in their study on street-vended pineapples, pawpaw, watermelons, and coconut. Similar research conducted in Jimma, Ethiopia reported that predominant bacteria isolated from the fruit juices were Klebsiella, Serratia, and Enterobacter (Tsige et al, 2008). [1] Another study conducted in India showed that pathogenic E. coli was seen in 27.7%, Shigella in 16.6%, Salmonella in 38.8%, and S. faecalis in 6.2% of the samples. [24] Journal of Pharmacy Research Vol 12 Issue

6 Antimicrobial Susceptibility Patterns The study was also focused on addressing antimicrobial susceptibility testing by means of a disc diffusion method on Mueller-Hinton Agar. The results of the antibiotic sensitivity test were interpreted and are presented as the resistant of bacterial isolates to the antibiotics [Table 5]. Most isolates were susceptible to ampicillin, gentamicin, ciprofloxacin, and chloramphenicol. All isolates were resistance to erythromycin, and most isolates were resistance to ciprofloxacin, amox-clavul acid, ceftriaxone, and tetracycline. According to the finding, erythromycin was not active against all bacterial isolates. All isolates of S. aureus were resistance to erythromycin and amoxclavul acid. 17.6% and 58.8 of isolates were resistance to tetracycline and ciprofloxacin, respectively, 41.1% and 64.7% to gentamicin and chloramphenicol, respectively. All isolates were sensitive to penicillin and cotrimoxazole. High rates of drug resistance were observed for Staphylococcus spp. against ampicillin (93%) and amoxicillin (92%). [25] Some E. coli isolates were resistant to amox-clavul acid, cotrimoxazole, ampicillin, and gentamicin. 50% were resistant to chloramphenicol and 83.3% were resistance to Ampicillin. Srinu et al. (2013) [26] also reported that E. coli was sensitive to streptomycin. In general, the findings from this study clearly indicate the poor hygienic conditions of these juices and the consumers are at risk of getting food-borne infections. Based on these data of the assed fruit juices, mango was found to be heavily contaminated with bacteria that could pose health problems. Lack of training (orientation) on food hygiene and safety; improper storage and processing of fruit juices may attribute to contamination of fruit during harvesting or poor processing and handling of fruit juices. The fruit juices investigated in this study had higher microbial load than the specifications set for fruit juices in some parts of the world. Based on the gulf standards, it is clear that the colony counts of the microbial groups in these fruit juices exceeded the standard. These high counts, however, may pose hazard to the health of consumers, especially if pathogenic species are present in the fruit juices to be consumed. They were also unaware of food regulations as well as lacking supportive services such as water supply of good and adequate quality, waste disposal systems that enhance their ability to provide safe food. In addition to, these the intensive and incorrect use of antimicrobial agents leads to the emergency of antimicrobial-resistant bacteria. CONCLUSION In general, the findings from this study clearly indicate the poor hygienic conditions of these juices and the consumers are at risk of getting food-borne infections. Based on these data of the assed fruit juices, mango was found to be heavily contaminated with bacteria that could pose health problems. Lack of training (orientation) on food hygiene and safety improper storage and processing of fruit juices may attribute to contamination of fruit during harvesting or poor processing and handling of fruit juices. The fruit juices Table 4: Frequency of Salmonella spp., S. aureus spp., E. coli spp., and Shigella spp. in avocado juice, mango juice, papaya juice, and Guava (n=45) Bacterial isolates Frequency (%) Avocado juice Mango juice Papaya Guava Total (%) Salmonella spp. 3 (6.6) 3 (6.6) 2 (4) 1 (2) 9 20 Shigella spp. 3 (6.6) 3 (6.6) 3 (6.6) 2 (4) S. aureus 5 (11) 4 (8.8) 4 (8.8) 3 (6.6) E. coli 4 (8.8) 2 (4) 1 (2) 2 (4) 9 20 Total (%) 15 (33.3) 12 (26.6) 10 (22.2) 9 (20) S. aureus: Staphylococcus aureus, E. coli: Escherichia coli Table 5: Antimicrobial susceptibility patterns of isolated bacteria from avocado juice, mango juice, papaya juice, and Guava juice in Axum, Ethiopia Antimicrobial drugs tested Bacteria isolates E. coli spp. n=6 (%) Shigella spp. n=8 (%) S. aureus spp. n=17 (%) R I S R I S R I S Ciprofloxacin (5 µg) Ceftriaxone (30 µg) Gentamicin (10 µg) Cotrimoxazole (25 µg) Erythromycin (15 µg) Amox clavul acid (30 µg) Ampicillin (10 µg) Chloramphenicol (30 μg) Tetracycline (25 µg) R: Resistant, I: Intermediate, S: Sensitive, S. aureus: Staphylococcus aureus, E. coli: Escherichia coli 514 Journal of Pharmacy Research Vol 12 Issue

7 investigated in this study had higher microbial load than the specifications set for fruit juices in some parts of the world. Based on the gulf standards, it is clear that the colony counts of the microbial groups in these fruit juices exceeded the standard. These high counts, however, may pose hazard to the health of consumers, especially if pathogenic species are present in the fruit juices to be consumed. They were also unaware of food regulations as well as lacking supportive services such as water supply of good and adequate quality, waste disposal systems that enhance their ability to provide safe food. In addition to, these the intensive and incorrect use of antimicrobial agents leads to the emergency of antimicrobial-resistant bacteria. Regular monitoring of the quality of fruit juices for human consumption must be introduced to avoid any future bacterial pathogen outbreak. Control measures should be introduced during sales of products, and these include displaying juices in glass cabinets, washing hands at regular intervals, disallowing customers from picking up and returning products with bare hands and/or without thorough hand-washing procedures. There should, also, be regular training/retraining of handlers in all aspects of food hygiene and safety. ACKNOWLEDGMENTS The authors sincerely thank to Aksum University research and publication directorate for providing financial support for this research work. It is also our great pleasure to acknowledge Aider Referral Hospital, College of health science, Department of Microbiology in Mekelle University for their invaluable help in providing and allowing the lab set up and other facilities. We have also a great appreciation to Mr. Areaya (Assistant professor) and Mr. Hayelom (lab tech) in Aider Referral Hospital for their meticulous assistance throughout the work. REFERENCES 1. Tsige K, Tsegaye G, Ketema B. 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