The relationship between human beings, food and microorganisms is unavoidable. Food is a substance which consists of different types of components such as protein, carbohydrates, fat, and many other nutrients used in the body of an organism to sustain growth and vital processes and to provide energy (Kharel & Tamang, 2010). Nowadays, street foods have become popular among millions of people because of their easy access and relatively cheap prices. Street foods are cooked and/or sold by vendors in public spaces, valued for their distinctive flavor, convenience, and preservation of the nutritional value of traditional foods (Hanashiro et al., 2005). But most of the vendors who sell these types of foods dont have any formal education on the maintenance of hygienic conditions during preparation and serving of food items (Mensah et al., 2002). Street foods are contaminated from various sources such as utensils, knives, raw foodstuffs, flies that periodically land on the foods, vendors bare hand serving and occasional food handling by clients (Nicolas et al., 2007). In many instances, tap water is unavailable for hand and utensil washing at vending locations; such washing is typically performed in one or more buckets, often without soap. Toilets, waste disposal and refrigeration facilities are seldom provided. Consequently, garbage is therefore disposed of in proximity, fostering conditions that attract insects, shrews, and household rodents, which may transmit food borne pathogens. As a result, microbial population gets opportunities to contaminate these items and so food borne illness of microbial origin has become a major public health problem (Tambekar et al., 2009). Thus, potential health risks are linked to food contamination by Escherichia coli, Salmonella typhi, Pseudomonas spp., Staphylococcus aureus, Proteus spp. and other species during preparation, and handling phases (Ghosh et al., 2007) (Hanashiro et al., 2005).

In Bangladesh, the demand of street foods is high at divisional cities due to tastes and varieties of foods, easy accessibility, low price, attraction for the foods, and above all, needs of the street people. For people from diverse social classes and regions specially the poor people, Cities have become hubs for employment opportunities and modern living. They get engaged in various types of work like garment workers, day laborers, rickshaw pullers, hawkers, vendors, etc. These people during their break time or mealtimes cannot eat at restaurants because they are too expensive. So, they must rely on street food, which is both affordable and appealing to them. Besides the students, people from the middle-class also visit   on weekends for street food, drawn by the attractive appearance of the food items (Khairuzzaman et al., 2014). Street foods, often prepared, processed and handled quickly and casually by non-professional personnel in developing countries, like in Bangladesh, are highly susceptible to microbial contamination from the surrounding environment (Mrityunjoy et al., 2013). In Bangladesh, popular street food items include fuchka, chotpoti, pitha, jhalmuri, singara, bhorta and many more. Most of these items are displayed in open spaces, likely to attract consumers quickly, but they are often stored improperly and lack appropriate protective packs (Noor, 2016). Indeed, the Bangladeshi people are highly susceptible to microbial attack due to densely populated environment with extremely unhygienic sanitary facilities.  This leads to the propagation of various food borne microorganisms into the surrounding environment in proximity and contributes to the prevalence of enteric diseases (Alam et al., 2015). Approximately, 30 million people in Bangladesh are affected by food borne diseases each year. Popular street food items in the country include chotpoti, fuchka, jhalmuri, velpuri, piyaju, puri, samosa, singara, beguni, chop and many others (Rahman et al., 2014; Rahman et al., 2019).

Microorganism-induced food borne diseases are a significant national and global health concern and a leading cause of mortality in developing countries (Garode & Waghode, 2012). Consumers have been reported to suffer from foodborne diseases like diarrhea, cholera, and food poisoning (Muzaffar et al., 2009). It has been reported that around 600 million people fall ill after eating contaminated street food and 420000 people die every year due to food poisoning (Morya et al., 2020). However, antimicrobial resistance among food borne pathogens has become more common in recent decades. According to a study 15.18% of S. aureus stains were found from street foods were resistant to methicillin. Every isolated bacterial colony exhibited resistance to Penicillin G. The methicillin-resistant bacterial colonies also exhibited resistance to kanamycin, gentamicin, tobramycin and erythromycin (Sina et al., 2011). In a similar study, the isolated Shigella species also observed multiple drug-resistant patterns (Oladipo & Adejumobi, 2010). Thus, this incidence of multidrug resistance ability among bacterial population is increasing day by day which is a thunder shock for human beings.

So, in our study, we focused on the evaluation of microbiological quality along with antimicrobial resistance pattern of isolates from street food items of higher educational institutions of Sylhet city. We also wanted to assess the knowledge of food hygiene of street food venders.

Selection of study location

For this study, five higher educational institutions of Sylhet city were selected as particular locations and selected institutes were Shahjalal University of Science and Technology (SUST), Sylhet Agricultural University (SAU), Leading University (LU), 

Metropolitan University (MU) and Murari Chand (MC) College (Fig. 1). For the present study, Street food vendors were defined as the persons who vended a variety of local food items among not only to the students at universities but also to people of all ages came there. So, samples were collected, and surveys were performed at different sites of universities which include approximately 50 m of distance from the entry gate of university and also from those vendors who were always available within the selected areas and agreed to provide sample for laboratory analysis. Thus, several zones of a university were selected for sampling and performing surveys.

Fig. 1: Location of five higher educational institutions of Sylhet city (https/www.sylhetdiv.gov.bd).

Preparation of questionnaire for survey and data collection

A short-structured survey questionnaire was prepared and pre-tested between 2nd January to 5th January 2021. With some modifications, the final questionnaire was organized in different sections with a total of 28 questions including mainly about: food venders basic information, information on his occupation along with his management of food preparation and serving, knowledge on hygiene practice and lastly knowledge of vendors on food safety. It was a face-to-face interview where data was collected according to the response of the vender whose interview was taken after proper observations of everything. The interview duration was about 20 min for each vendor. The interviews were conducted from 7th January to 11th January 2021. Each question was asked systematically in a very simple manner with explanations wherever necessary. Each interview was conducted only after the consent given by the respondent food venders to participate in the interview. The questionnaire didnt include anything which might cause harm to their occupation.

Collection of samples and sterilization of glassware and media 

Street food items were collected from different food venders of Sylhet city from 7th January to 11th January 2021. Five types of street food items (Fuchka, Chotpoti, Singara, Piyaju and Puri) were collected from MU, MC College, SAU, SUST and LU respectively. These samples were kept within sterile plastic containers and carried to the laboratory of the Faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University, Sylhet using a sample collector ice box where temperature was maintained at 4°C so that samples didnt get spoiled. Finally, the box was transported to the laboratory without delay and preserved in a refrigerator at 0-4°C until examination but not longer than 36 hours. All the media were sterilized using autoclave at 121˚C temperature at 15 psi pressure for 15 minutes before use. All the glassware (such as Petri dishes, test tubes, pipettes etc.) were made sterile at 180˚C temperature for 1 h (Pisano et al., 1966).

Processing of samples

After collecting samples, these were processed before isolation of bacteria. At first, samples were taken out from the refrigerator and were analyzed after reaching room temperature. About 10 gm of each sample was homogenized using measured volume (90ml) of 0.1% buffered peptone water with the help of mortar and pestle. A serial dilution was performed using sterile peptone water. The homogenate sample was first diluted at 1:10 ratio, and subsequent dilutions were prepared by adding 1ml of homogenate into 9ml of buffered peptone water (Tang et al., 2018). Finally serial dilutions of samples were made up to 10-8 in sterile peptone water for total viable bacterial count (TVBC), total coliform count (TCC) and total staphylococcal count (TSC).

Isolation and enumeration of bacterial colonies

Spread plate technique was followed to isolate and count bacterial population after performing serial dilution. 0.1 ml of samples from each dilution were taken onto sterile petri plate and spread evenly on three types of culture media namely Nutrient Agar, MacConkey Agar and Mannitol Salt Agar. Then all the Petri plates were incubated at 37˚C temperature for 24-48 hrs. Nutrient Agar, MacConkey Agar and Mannitol Salt Agar were used for performing total viable bacterial count (TVBC), total coliform count (TCC) and total staphylococcal count (TSC), respectively in cfu/ml (Adimasu et al., 2016).

Morphological and biochemical identification of bacteria

Phenotypic characterization of bacterial isolates was conducted using gram staining technique. This procedure differentiates between Gram positive and Gram-negative groups by coloring these cells red or violet respectively. Additionally, bacterial morpho-logy was identified using microscope following the staining procedure (McClelland, 2001). Several biochemical tests, including oxidase test, catalase test, indole test, citrate test, MR-VP test, triple sugar iron test, were performed to identify bacterial species based on variations in their biochemical activities (Senthilkumar et al., 2014).

Screening of multidrug resistant bacterial isolates

Multidrug resistant bacteria were screened using Kirby-Bauer disc diffusion method on Mueller-Hinton agar, following the guidelines (Bauer et al., 1966; Clinical & Laboratory Standards, 2019). Total 8 antibiotics used in this study were ampicillin, gentamicin, erythromycin, cefepime, ceftazidime, ceftriaxone, cefotaxime and chloramphenicol. At first, inoculums were prepared using nutrient broth. Then from EMB and TCBS agar plates, single colonies were transferred into test tubes containing 10 ml of broth and incubated at 37°C temperature for 18-24 hrs. until attaining proper growth. Mueller-Hinton agar was prepared as per instructions provided by the manufacturer. Around 100 μl of bacterial culture was taken from broth using a micro-pipette and spread in the plate containing Mueller-Hinton agar with sterile cotton bud. Antibiotic discs were placed aseptically into center of agar plates with the help of a sterile forceps and incubated at 37°C for 16-18 hrs. The zone of inhibition was observed to assess antibiotic sensitivity or resistance, and the zone diameter was measured using a ruler. 

Basic information on food vendors

A survey on the pre-structured questionnaire was carried out among 25 food vendors. It was found that 96% of vendors were male among 25 vendors and all the food vendors were literate at their level. 52% of them had educational qualifications within primary level and it was maximum percentage. 44% of them had experience of vending for 7-9 years according to the report. The maximum of them had daily income within the range of 500-1500 tk. 0nly 3% had daily income between 3501-4500. 56% of the vendors took this business as their principal way of livelihood according to the investigation (Table 1).

Table 1: Food vendors basic information.

Information about food vending

According to the report, chotpoti and fuchka were most frequently sold items. It was found that among different types of food items, the highest selling of items was fuchka and chotpoti and it was 28%. After fuchka and chotpoti, the second most frequent selling items were singara and chop which attained 24% of total percentage. Most of the food vendors preferred to sit for sale during the evening. In this investi-gation, it has been found that 32% of the vendors sold food from 6 PM-12 PM while 28% preferred to sell food from 3 PM-8 PM. Though people of all ages love street foods, here 60% of the consumers were underage of 20-25 years as reported while 28% and 12% consumers were under the age of 26-30 and 31-35 respectively. Among different food items, demand for fuchka was highest (Table 2)

Table 2: Information on food vending.

Sanitation and hygiene practices

Sanitation refers to access to facilities for the safe disposal of human waste (feces and urine) including behaviors, and services that prevent diseases caused by contact with human waste. In contrast, hygiene pertains to practices that enhance cleanliness and promote good health. Proper hygiene acts as a barrier against many infectious diseases, including fecal–oral diseases, thereby supporting improved health and well-being (Muyanja et al., 2011). Street food vendors responded to the above-mentioned variables as either “Yes” or “No” and their answer was marked as either “1” or “0” respectively. The total score of the variables were determined within ranges from 0 to 8 and venders scoring 7or 8 were marked as good hygiene maintainer though there was not found at least one vendor who followed all the hygienic rules. Their responses were used to create a score list, where the score 1 indicated the highest risk of health hazards. 28% of them got 4 marks and 16% achieved 7 marks for good hygiene practice. 24% of the vendors scored 3 that indicated remarkably too bad condition of hygiene (Table 3 and 4). 

Table 3: Sanitation and hygiene practices.

Table 4: Scoring of good hygiene practices.

Fig. 2: Representation of column chart showing vendors management of food preparation and serving.

Knowledge of food vendors on food related issues

It is important for all the vendors to have some knowledge on how to keep foods safe and fresh along with the information on food-borne diseases so that they can take appropriate initiatives to protect food items from contamination which will also help them to seek attention from consumers and thus the vendors can attain popularity for their good main-tenance of hygienic condition. According to this investigation, this type of knowledge among food vendors was not up to the mark as most of them only have primary level education. Fig. 6 showed that 80% of food vendors didnt ensure food safety during transportation as they used only paper bags to deliver food. 20% of them packaged foods in foil bags for transportation of foods away from the selling place. 92% of vendors had little knowledge about food borne diseases and pathogens related to food poisoning (Fig. 3). Moreover, none of them received any training related to food safety and so they didnt know how to keep foods contamination free and what could be the worst scenario of food poisoning.

Fig. 3: Representation of column chart showing knowledge of food vendors on food related issues.

Isolation and enumeration of bacterial colonies

In this study, 5 types of different street food samples (piyaju, singara, puri, fuchka, chotpoti) were collected from 5 higher educational institutions of Sylhet city (Table 5). 

 

Table 5: Enumeration of bacterial colonies in tested street food samples.

From Table 5, it was noticed that the highest total viable bacterial count (2.4 x 109 cfu/ml) was found in sample fuchka, collected from Metropolitan University (MU) and lowest total bacterial count was 2.2 x 108 cfu/ml, which had been found in singara collected from SAU. The highest coliform bacterial count was (5.5 x 106 cfu/ml) found in Puri which was collected from LU and lowest total coliform count was (4.6 x 103) found in singara collected from SAU. The highest staphylococcal count was (2.94 x 106) found in piyaju collected from SUST and the lowest staphylococcal count was found in (3.0 x 104 cfu/ml) singara collected from SAU. 47 isolates were randomly selected from Nutrient Agar, MacConkey Agar and Mannitol Salt Agar media. The morpholo-gical identification of these selected bacterial isolates was confirmed through gram staining test and biochemical tests (indole test, citrate test, oxidase test, catalase test, methyl red tesr, voges-proskauer test and TSI test) (Fig. 4). The test results revealed that among 47 bacterial isolates, there were 4.25% Vibrio cholerae, 6.38% Proteus mirabilis, 25.53% Klebsiella spp., 17.02% E. coli, 8.51% Pseudomonas spp, 38.29% Staphylococcus aureus (Fig. 5).

Fig. 4: Colonies of bacteria after plating (A) TVBC, (B) TCC, (C) TSC.

Fig. 5: Percentage of bacterial isolates in ready-to-eat food samples.

Antibiotic susceptibility profile of bacterial isolates

Total 8 antibiotics were selected based on their mode of action (inhibitor of cell wall synthesis, protein synthesis, cell division and nucleic acid synthesis) and rank of generation (1st, 2nd, 3rd and 4th generation) and tested against 16 isolates selected randomly (Fig. 6). All the tested isolates showed resistance against ampicillin and cefepime according to the guidelines of CLSI (Clinical and Laboratory Standards Institute) standards. Resistant isolations against gentamicin, erythromycin, ceftazidime, ceftriaxone, cefotaxime and chloramphenicol were 31.25%, 25%, 25%, 93.75%, 12.5%, 25%, 25%, 25%, 25%, 25%, 18.75%, respectively (Table 6 and Fig. 7).

Fig. 6: Antibiogram of bacterial isolates.

Table 6: Percentage of antibiotic susceptible, intermediate and resistant bacteria.

Fig. 7: Representation of column chart showing antibiotic susceptibility profile of bacterial isolates.

This study was conducted with a view to represen-ting potential food safety threat associated with street foods collected from the higher educational institutions of Sylhet city along with the assessment of food hygiene knowledge of food vendors. A short, structured survey showed that males made 96% of the vendors whose interviews were taken and only 4% sector was for female (Table 1). In contrast a study reported the participation of 47% female vendors in their study conducted in Kenya (Bereda et al., 2016; Gitahi, 2012). Among 25 vendors, 52% had primary education; 40% had secondary education and 8% had higher secondary education. Among different items, fuchka was the most popular one according to the 40% of vendors (Table 2).

When handling and cooking street foods, hygiene is crucial.  Around 52% of them didnt use any kind of soap or detergent to wash dirty utensils (Fig. 5). They didnt even change water and reuse it to wash dishes. In a study conducted by Tesfaye et al. (2016) (Bereda et al., 2016) in Eastern Ethiopia, it was observed that 39.4% washed their utensils with dirty water that was reused multiple times. Moreover, 64% of the food vendors made the food under the open sky and in the open space which made it easier for the microorganisms to build up their habitats in the food (Fig. 5). According to by Oranusi et al. in Nigeria, (2012) (US & Braide, 2012) roughly 85% of the vendors prepared their foods in unsanitary settings. This is also true in our study where 84% of vendors disregarded hygiene regulations (Table 4). Conversely (Von Holy & Makhoane, 2006) found that street food vendors could provide safe food with low bacterial counts, but there was still a need for appropriate hygienic circumstances and access to basic sanitary facilities. This might be possible because the vendors were educated and received basic professional training in food safety. According to the current study, it has been found that 92% of food vendors used to offer meals without wearing gloves (Fig. 5). This finding is also related with the findings of Tambekar et al. (2011) (Tambekar et al., 2011) in India who also found the practice of using bare hands of vendors during handling of foods and the percentage was 47.62%. In this study it was reported that only 92% of the vendors had knowledge of food borne diseases. However (Martins, 2006) his study demonstrated that a high hygiene standard was maintained by most vendors (85%) during preparation and serving, attributed to their knowledge on food safety. However, this was not observed in our study.

Nearly all the street food samples had a high microbial load.  According to FAO (1997) (Food & Agriculture, 2007), the standard limit for total microbial counts in street food has ranges from 1x104 cfu/ml to 3x104 cfu/ml. In comparison, the bacteriological quality of most street food samples in this study fell significantly below the standard level (Table 5). The study revealed that the highest total viable bacterial count (TVBC) was 2.4x109 cfu/ml. Moreover, the lowest TVBC was 2.2 x 108 cfu/ml, which also crossed the satisfactory level. It may result from unhygienic conditions of the environment where food is prepared and served. This TVBC has close links to the research conducted in Dhaka, Bangladesh by (Mahfuza et al., 2016). They also examined salad and discovered that this range varied between 3.9x105 and 4.3x106 cfu/ml. This reflects the poor standard of street food hygiene maintenance.

In this study, the level of coliforms (TCC) varied from 4.6 x 103 cfu/ml-5.5 x 106 cfu/ml (Table 5) which crossed the standard level (<103 cfu/ml) (Choudhury et al., 2011). Vendors often prepare and serve the food in bare and unwashed hands, which is likely one of the primary sources of contamination (Khairuzzaman et al., 2014). According to a study of Susmita et al. (2020), the range of TCC in street food of Noakhali was 1.55×104 - 4.05×105 cfu/ml which was strongly linked with our finding range (Ghosh et al., 2020). According to Asefa et al. (2016), the level of coliforms in street food of Gondar town in Northwest Ethiopia varied between 0.3-1.2×104 cfu/ml which showed association with our result (Adimasu et al., 2016).

Again, Total Staphylococcal Count (TSC) of this study was calculated within the range from 3.0 x 104 cfu/ml-2.94 x 106 cfu/ml (Table 5) which was higher than standard level (1.0 x 103 cfu/ml) [31]. A study conducted by Eromo et al. (2016), found TSC as range between 1.7×105 cfu/ml – 2.2×105 cfu/ml which showed a connection with our study (Eromo et al., 2016). This type of contamination could be linked to inadequate personal hygiene and the vendors contaminated hands, as the bacterium is typically associated with human skin and clothing. Finding of this study is also connected with another study conducted by Beatrice et al. (2020) in Kenya where TSC was counted within range of 1.5 ×104 cfu/ml- 3.3×105 cfu/ml (Birgen et al., 2020).

In this study, it was predicted that there were 38.29% Staphylococcus aureus and 17.02% E. coli in the tested samples after biochemical tests (Fig. 5). Suneetha et al. (2017) also reported the presence of S. aureus in street food samples of Chennai, India. S. aureus can cause pathogenic diseases to humans through the production of toxins (Suneetha et al., 2011). The production of an effective level of toxin requires a high concentration of microorganisms (approximately 105 -106 microbes per ml of food (Howden et al., 2010). This amount was available in our tested samples. The presence of large number of E. coli was also noticed in this study (Fig. 5). Prevalence of E. coli might occur due to post-processing contamination and fecal contamination as food vendors didnt have the facilities for maintaining proper sanitation practices. E. coli is a common bacterium in stool and the environment and is the main member of the facultative anaerobic microbiota of the intestines., Nobili et al. (2017) in Italy also found presence of E. coli in their tested samples which is associated with this report. Serious illnesses are all known to be brought on by pathogenic strains (Nobili et al., 2017). Among gram-negative coliforms, E. coli is the most prevalent pathogen responsible for meningitis particularly during the neonatal period. It is associated with significant morbidity and mortality rates globally. The incidence of travelers diarrhea sometimes converts into a life-threatening state due to the presence of E. coli in food samples (Ekici & Dümen, 2019). In our study, the presence of E. coli in street food samples thus indicates serious threat to the consumers. In this study, all the test results also revealed the presence of 4.25% Vibrio cholerae, 6.38% Proteus mirabilis, 25.53% Klebsiella spp., 8.51% Pseudomonas spp. besides S. aureus and E. coli. The indication of V. cholerae is an alarming fact for the consumers. John et al. (2018) in Japan also found the presence of V. cholerae in street food samples which can cause severe intestinal infection which is related to our finding (Tang et al., 2018).

A total of 8 antibiotics were tested against selected 16 isolates chosen at random to determine antibiotic susceptibility. All the tested isolates showed resistance against ampicillin and cefepime. Resistant isolations against Gentamicin, Erythromycin, Ceftazidime, Ceftriaxone, Cefotaxime and Chloramphenicol were 31.25%, 25%, 25%, 25%, 93.75%, 12.5%, 25%, 25%, 18.75%, respectively (Table 6) which also included multidrug resistant S. aureus and E. coli. Our study showed similar results with the study of Tabassum et al. (2015) where it was reported that both velpuri and water of velpuri were contaminated with multidrug resistant Enterobacter spp (Tabassum et al., 2015).

The primary limitation of this survey is small data size, which prevents us from confidently concluding that our findings represent the exact scenario for all types of street food items. But through all the observations, it can be said that due to the lack of educational facilities and training system, microbial contamination of street food items is getting worse day by day.

Food is the source of energy in the human body. Day by day, the demand for street food is increasing among people of all ages. But unhygienic handling of these food items is causing various diseases. This study also confirmed the considerable rate of microbial contamination in street vended foods due to the lack of knowledge on food security and safety of street food vendors. The identified foodborne bacteria and antibiotic-resistant isolates can pose a serious public health problem. Therefore, regular inspection and education of vendors on food safety and hygienic practices are required to improve health standards of consumers.

The investigation, conceptualization, writing-reviews, supervision was conducted by M.A.M, writing-original draft, data collection, and laboratory experiments were done by A.D., A.G., and M.R.A, writing-critical reviews, editing, data analysis, and references were prepared by S.K.S. All authors have read and agreed to the published version of the manuscript.

This article is original work and the authors declare that there is no conflicts of interest relevant to this article.

The study did not involve the use of animal models or human participants. This study was conducted in compliance with Sylhet Agricultural University ethical standards and guidelines. All experimental procedures were carried out in accordance with institutional and international ethical standards for laboratory-based research.

We thank the faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University, Sylhet for the laboratory support.