Bacterial load Assessment of some Food items sold in street in Woldia town, North-east ethiopia

Infectious diseases due to contaminated street vended food items has happened to an essential concern and public health issue in low socio-economic status individuals. The goal of this study was to evaluate bacterial quality and safety of some food items sold in street based on aerobic mesophilic bacteria, Salmonella, Staphylococcus aureus and Escherichia coli in Woldia town. Cross-sectional study was performed on some selected street vended foods from March 2019 to May 2019 in Woldia town. A total of 36 ready-to-eat food samples such as ‘Sambussa’, ‘Bonbolino’ and ‘Ambasha’ were collected for microbial analysis. Finding of the study indicates that 32(88.9 %) of the vendors were females, 28(77.7%) handled food with bare hands, 28(77.7%) vendors were washing their hands after toilet. The mean total count of aerobic mesophilic bacteria, Salmonella, Staphylococcus aureus and Escherichia coli was 4.5x102±5.2x101 CFUg-1, 1.5x102±4.5x101 CFUg-1, 1.1x102±1.4x101 CFUg-1 and 2x102±3.4x101 CFUg-1, respectively. The dominant bacterial isolate from the street vended foods was Escherichia coli (53.5%), followed by Salmonella (24.1%) and Staphylococcus aureus (22.4%). The result of this study demonstrates the non hygienic practices of the vendors in street vended foods showed high bacterial load. Therefore, the street vended foods were contaminated with food borne pathogenic bacteria that can create a potential risk to public health in the study area. In general, the bacterial quality of the food quality sold in street in Woldia town was very poor and needs especial attention to avoid bacterial contamination.


iNtROduCtiON
In low income countries foods sold in the street by food vendors prepared at home and consumed on the street without further preparation 1 . In developing countries where there is high unemployment, low salary incomes, little work chance and where urbanization is taking place, consumption of street vended food items is common 2 . Both food venders and consumers are beneficiary from the street vended foods. Street food items are accessible, cheap, and nutritionally-balance and also supply a base of income, opportunity of self-employment and chance to develop business skill with low capital investments to the vendors 3 . Foods vended in the street are predisposed to contaminations since they are exposed to dust and are often not covered. Consuming unsafe food poses an important community health risk in both developed and developing countries 4 . Diseases due to contaminated food are still a common problem and causes mortality and morbidity in the community. Transmission of food related diseases due to lack of hygiene of food handlers is a common worldwide problem and a wide range of enteric pathogenic microorganisms might be carried by food handlers and implicate infections in the public 5 . A number of scholars reported that food borne infections have been alarmingly increasing throughout the world. These reports have shown that inadequate food hygiene and safety practices during food handling, preparation and serving are the major causes 6 . Even if there is a well awareness about diseases transmitted because of consumption of contaminated street vended food items, most of the consumers do not give attention these health hazards 7 . Several studies have showed that different types of disease causing microorganisms such as Bacillus cereus, Staphylococcus aureus, Salmonella spp and E. coli have been obtained in various types foods sold in the street 8 . Every year, food borne infections cause the suffering of millions of people throughout the world 9 . A report done in 2005 only showed that diarrheal diseases cause for the death of 1.8 million 10 . The National Hygiene and Sanitation Strategy program in Ethiopia reported that about 60% of the infection load is related to poor hygiene and sanitation practice of the consumers 11 . Salmonella, Shigella and related food borne infectious causing microorganisms were identified in the foods sold in the street and become serious health problem in Ethiopia. Diarrheal disease represents the second leading cause of death in Ethiopia 12,13 . Uses of street foods are nowadays common in different towns of Ethiopia and it is observed in areas of many people found such as bus stations, schools and other places. The low income category of the people commonly consumes foods sold in the street in Woldia town. However, the awareness of the consumers on the safety and microbial load of the food items sold in the street and related factors in this study area are limited.
The main purpose of the present study was to investigate the microbial safety of street vended foods and food handling practice of the venders in Woldia town, North East Ethiopia.

MATERIALS AND METHODS The study area
This study was done in Woldia, the main town of North Wollo, located 530km North East of Addis Ababa, the capital city of Ethiopia (Fig. 1). The town's geographical coordination is 11°50′N latitude and 39°36′E longitude. The town has an elevation of 2112 m above sea level.

Study design
A cross sectional study design was conducted in Woldia town from February to June 2019 to examine the bacteriological quality and handling practices of food sold in the street such as 'Sambussa', 'Bonbolino' and 'Ambasha' vended at Gonderber, Mugad, Menehariya and Piazza. In the present study the study population was randomly selected street food venders in Woldia town.

Sampling technique
Random and nonrandom sampling techniques were used to select representative street food sellers in the study area. Four common street food vending sites such as Gonderber, Mugad, Menehariya and Piazza were selected purposefully. A Total of 36 samples of food items sold in the street which consist of 12'Sambussa', 12 'Bonbolino' and 12'Ambasha' were selected by random sampling technique and taken into microbiology laboratory of Woldia University for bacteriological load analysis.

Socio-demographic data collection
Socio-demographic data and personal hygiene practices of the street food vendors from 36 venders were selected by using random sampling technique. The data were collected by face to face interview method using semistructured questionnaire and an observation checklist.

Sample collection
Three most street vended food stuffs, namely 'Sambussa' 'Bonbolino' and 'Ambasha' were selected for bacteriological analysis and randomly bought from street food vendors. The food items for laboratory analysis were aseptically collected from street food sellers by using sterile glass material and immediately stored in icebox. All the collected food items were taken to microbiology laboratory of Woldia University for bacteriological analysis. The collected food samples were placed in the refrigerator at 4°C till bacteriological analysis was carried out. Bacterial analysis was conducted within one to three hours after sample collection.

Bacteriological methods Food sample preparation
For microbiological analysis 10g of each food samples namely 'Sambussa', 'Bonbolino' and 'Ambasha' were homogenized in 90 ml buffered peptone water. Then after, the food sample homogenates were processed to serial dilutions of 10 -2 , 10 -3 , 10 -4, and10 -5 of all food items, the suspected bacterial load of 0.1ml dilutions were transferred to plates and spread by L-shaped glass tube to plates and incubated at 37°C for 48 hours, finally the load of bacteria counted by colony counter. The counted colonies were expressed in colony forming units per gram (CFUg -1 ).

Microbial enumeration Aerobic mesophilic bacteria
The pour plate technique on plate count agar (PCA) (Oxoid Ltd, United Kingdom) was used to count aerobic mesophilic bacteria. A homogenized food sample of 0.1ml was inoculated onto plate count agar in triplicate and the plates were incubated aerobically at 37°C for 48 hrs. All plates showing colony counts between 30 and 300 were selected and their colony forming unit per gram of food item (CFUg -1 ) were calculated by multiplying by the dilution factor.

Isolation of Salmonella
To detect Salmonella, 10g of food sample was homogenized in 90 ml buffered peptone water and shook for 2-3 minutes using shaker (STUART, UK) to prepare homogenate dilution. A serial dilutions of 10 -2 , 10 -3 , 10 -4, and10 -5 were underwent from homogenate food sample and 0.1ml dilutions were transferred to xylose-lysine-deoxycholate (XLD) agar and spread by L-shaped glass tube to plates and incubated at 37°C for 48 hours. Then black centered pink colonies were enumerated using colony counter (STUART SCIENTIFIC, UK). Colony forming unit per gram of food item (CFUg -1 ) was determined by multiplying with the dilution factor.

Isolation of Staphylococcus aureus
For Staphylococcus aureus, 10g of food sample was homogenized in 90 ml buffered peptone water and shook for 2-3 minutes using shaker (STUART, UK) to prepare homogenate dilution. A serial dilutions of 10 -2 , 10 -3 , 10 -4, and10 -5 were underwent from homogenate food sample and 0.1ml dilutions were transferred to sterile molten Mannitol salt agar (BLULUX) petri plate and then incubated at 37°C for 48 hours. Finally, due to Mannitol fermentation yellow to orange colonies surrounded by yellow zone was enumerated using colony counter (STUART SCIENTIFIC, UK) and the obtained data was recorded as colony forming unit per gram (CFUg -1 ) of all food items.

Isolation of E. coli
Ten gram of food sample was homogenized in 90ml buffered peptone water and shook for 2-3 minutes using shaker (STUART, UK) to prepare homogenate dilution. A serial dilutions of 10 -2 , 10 -3 , 10 -4, and10 -5 were underwent from homogenate food sample and 0.1ml dilutions were transferred to Eosin Methylene Blue (EMB, Himedia, India) agar for isolation of E. coli and spread by L-shaped glass tube to plates and incubated at 37°C for 48 hours then green metallic sheen colonies showing a dark nucleated centre were recorded using colony counter (STUART SCIENTIFIC, UK).

Data analysis
Data obtained from different food samples were analyzed by using SPSS software version 20.0 and mean bacterial loads of food samples from different sites were compared using one way ANOVA. A p value of ≤ 0.05 at 95% confidence interval was considered as statistically significant.

Knowledge about food safety and personal hygiene of the food sellers
As indicated in table 2, 28(77.8%) of the street food sellers had a good awareness of food safety, while 8(22.2%) of the street food sellers had no awareness of food safety. This implies that the vendors contaminate the food items during preparation, handling, transportation, and while they sold. All vendors, 36(100%), knew hand washing before work reduces the risk of food contamination as well as proper utilization of utensils, proper handling ,well cooking of food,   14 . In all vending site no clean water at the site or close to the site, no washing facilities for hands, vendors didn't wash down their hands in clean water each time before the handling and serving of food and all (100%) of the street food vendors didn't wash hands after handling money before handling food again and 30(83%) of the vendors handle food or money with uncovered hands.

Bacteriological analysis of street-vended foods
From four street food vending sites 36 food samples were analyzed from the food items to assess the level of contamination based on aerobic mesophilc bacteria, Salmonella, Staphylococcus aureus and E.coli is indicated in table 3. The study showed that the food samples were contaminated by pathogenic bacteria. Three types of bacterial species were identified from the three kinds of food items. The maximum numbers of Salmonella was seen in 'Sambusa' (S1) from Gonderber and the maximum numbers of S.aureus was observed in 'Bonbolino' (S31) from Piazza. In the case of E. coli the highest load was observed in 'Bonbolino' (S13) from Mugad. A total of 18694 bacterial isolates were analyzed from 36 food samples, 5437 bacterial strains were isolated from 'Sambussa', 7321 from 'Bonbolino' and 5936 from 'Ambasha' by using selective media. Among the total bacterial isolates, the dominant bacterial group was E.coli (57%) followed by Staphylococcus aureus (23%) and Salmonella (20%).
In this study the socio-demographic characteristics indicated that the majority (89.9%) of street food sellers were females. A study done in Gondar, Ethiopia reported that 95% of the vendors were females 16 . Similarly a study done in Jima 17 also reported the majority (85.5%) of street food vendors were females. Among 36 venders participated in the current study, 28(77.8%) of the vendors were washing their hands after toilet which was high compared to findings in Hawassa, Ethiopia (68.6%) 5 and Gondar (63.3%) 18 but it is in line with Addis Ababa, Ethiopia (80.8%) 19 and Jimma, Ethiopia (77%) 20 . In this study 66.7% of the vendors cover their hair while serving and handling foods which was higher than a study reported in Ihiagwa (12.7%) 21 .
In the present study, the greater part (83%) of the vendors served their customers by handling food with bare hands. A similar study done in Jigjiga City, Ethiopia 4 indicated 47.62% of the street food sellers handled food with uncovered hands. The street food sellers can be contaminated by pathogens like Salmonella, S. aureus and E.coli and in due course can transmit these food borne disease causing microorganisms to the consumers. This is the fact that those street vended food items are frequently contacted with unhygienic hands of vendors at some point in preparation, handling and serving 22 . World Health Organization 23 reported those unhygienic food handlers' hands are the main significant vehicle for the transfer of microorganisms from skin, feces and nose to the food.
Overall study indicated that all the food items sold on the street were contaminated with variety of pathogenic microorganisms. As indicated in table 5, in this study, the mean total count of AMB was 5.2x10 2 ±3.4x10 1 CFUg -1 . The load of aerobic mesophilic bacteria is significantly high in 'Bonbolino' mainly due to unhygienic condition of the venders. A similar study in Yaounde-Cameroon 24 , reported there was high count of AMB in the street vended food items. Similarly, a study done in Ambo, Ethiopia 25 reported that the highest AMB count (2.6x10 3 CFUg -1 ) was observed in street vended foods. Another study done in Jima, Ethiopia 17 reported a mean aerobic mesophilic bacterial count of 10 5 ±3. 16 CFUg -1 which was higher than the present study. The mean total count of Salmonella in the study area was 3.1x10 2 ±1.2x10 1 CFUg -1 which was in agreement with a study done in Hawasa, Ethiopia 22 . In Ago-Iwoye, Nigeria 26 , reported only 0.7% of the food samples were contaminated with Salmonella which was very low compared with the present study.
In the case of Staphylococcus aureus, the mean count in the study area was 1.2x10 2 ±1.4x10 1 CFUg -1 . High mean count of Staphylococcus aureus (1.4x10 2 ±4.2x10 1 CFUg -1 ) was observed in Mugad. A study done in Bahir Dar, Ethiopia 3 observed high load of Staphylococcus aureus in 'Sambussa' which was in agreement with the findings of the present study. Similarly, a study done in Gonder 27 35.54% of the food samples were contaminated with Staphylococcus aureus. Another study done in Jima, Ethiopia 28 28.2% of the food samples were contaminated by Staphylococcus aureus which was higher than the frequency of the present study (23%).
The mean count of E.coli observed in the study area was 2.9x10 2 ±2.1x10 1 CFUg -1 which was higher than the mean count of AMB, Salmonella and S. aureus. High mean load of E.coli count was observed in Mugad (3.9x10 2 ±5.2x10 1 CFUg -1 ). The load of E. coli observed in the present study was not in line with a previous result reported in Gondar 29 . A study done in Bangladesh 30 a maximum of 4.0x10 3 CFUg -1 E.coli was reported that was not in agreement with the present findings. A similar investigation done in Zimbabwe 31 reported that 53% of the food samples were contaminated with E. coli which was lower than the present study. The occurrence of high mean count of E.coli in this study area demonstrates a potential high health risk and their occurrence is a suggestion of likely fecal contamination of food or water, water or food handlers and inferior hygienic processing practices 8 .
A high mean count of AMB, Salmonella, S. aureus and E.coli suggests contamination of foods due to inappropriate processing, contamination through contact with contaminated utensils and poor personal hygiene of the vendors.

CONClusiON
This study revealed the street vended food items were potentially contaminated by the Salmonella, S.aueres and E. coli. Therefore, the occurrence of these microorganisms might be predicted for the presence of potential disease causing microorganisms. Very high bacterial loads from 'Sambussa', 'Bonbolino' and 'Ambasha' food samples suggests a potential health risk to the consumers from the utilization of foods sold in the street in the study area. This indicates poor food handling practice and personal hygiene of the vendors in the street and high risk of food borne infections. To enhance bacterial quality of foods sold in the street by venders providing regular training and creating awareness on food handling and personal hygiene among street food venders as well as consumers is recommended.

ACKNOWLEDgMENTS
The author acknowledges the study participants and Woldia University for providing necessary facilities and infrastructure to perform the tests.

FuNdiNg
None.

ETHICS STATEMENT
Ethical clearance was obtained from the Ethical Committee of Woldia University. .

dAtA AvAilABility
All the necessary data are available in the submitted manuscript.