Isolation and identification of bacteria in different street vended foods collected from selected areas of Bangladesh

Street food vending has become an important public health issue and a great concern to everybody. This is due to widespread food borne diseases, due to the mushrooming of wayside food vendors who lack an adequate understanding of the basic food safety issues. Major sources contributing to microbial contamination are the place of preparation, utensils for cooking and serving, raw materials, time and temperature abuse of cooked foods and the personal hygiene of vendors. The objective of the study was to explore the microbiological quality of different street food; Chotpoti, Chanachur, Amra (Spondias mombin) and Jolpai (Elaeocarpus serratus (Veralu / Ceylon Olive) sold by various street vendor at Khulna city, Bangladesh. A total of 20 samples were collected randomly from street vendors and tested for the presence of bacteria following standard microbiological method used for isolation, enumaration and identification of bacreria. All the examined samples were contaminated by various types of bacteria. The total viable count (TVC) in different street food samples was ranged from 9.6 x 10 CFU/g to 5.9x10 CFU/g. Among samples Escherichia coli 8(40%), Staphylococcus spp. 5(25%), Klebsiella spp. 4(20%), Salmonella spp. 1(5%) and Shigella spp. 2 (10%) were isolated. Escherichia coli were found highest 3(50%) Jolpai samples, then another organisms Staphylococcus spp. was found highest 2(50%) in Chotpoti and then Klebsiella spp. was found one in each sample. Therefore, application of sound risk analysis policies is being advocated to provide a scientific base to the host of risk management option which Bangladesh may need to explore to ensure public health and safety.


Introduction
Street foods are defined as ready-to-eat foods and beverages prepared and/or sold by vendors and hawkers, especially in streets and other similar public places (Bereda et al., 2016). This industry plays an important role in meeting the food requirements of urban dwellers in many cities and towns of developing countries. It feeds millions of people daily with a wide variety of foods that are relatively cheap and easily accessible (Rane, 2011). However, food borne illnesses of microbial origin are a major health problem associated with street foods (Islam et al., 2015). Food contamination with antibiotic resistant bacteria can also be a major threat to public health, since the antibiotic resistance determinants can be transferred to other pathogenic bacteria potentially comprising the treatment of severe bacterial infections Sharma et al., 2015). The traditional processing methods that are used in preparation, inappropriate holding temperature and poor personal hygiene of food handlers are some of the main causes of contamination of street-vended foods.
Consumers who depend on such foods are more interested in its convenience and usually pay little attention to its safety, quality and hygiene (Muleta and Ashenafi, 2001). Ready-to-eat street foods are also subjected to cross-contamination from various sources such as utensils, knives, raw foodstuffs, flies that sporadically landing on the foods, vendors bare hand serving and occasional food handling by consumers (Muzaffar et al., 2009;Karmaker et al., 2018). In most cases, tap water is not available for washing hands and utensils at vending sites; hand and utensil washing are usually done in one or more buckets-sometimes without soap. Toilets, waste disposal and refrigeration facilities are rarely available (Hassan et al., 2018). Wastewater and garbage are therefore discarded nearby, providing nutrients for insects and other household rodents, which may carry food borne pathogens (Barro et al., 2006). Thus, potential health risks are associated with contamination of food by Escherichia coli, Salmonella typhi, Pseudomonas species, Staphylococcus aureus, Proteus species and other species during preparation, postcooking and various handling stages (Tambekar et al., 2008;Schmidt et al., 2003). As a result, food borne illness associated with the consumption of street foods has been reported in several places such as Bangladesh, India (Khairuzzaman et al., 2014), Mexico and Ethiopia. In Bangladesh, health risks associated with street foods are common. Salmonella, Shigella and other food-borne pathogens were identified in similar studies on street-vended foods in this country (Garode and Waghode, 2012). On the other hand, the prevalence of antimicrobial resistance among food borne pathogens has increased during recent decades (FAO and WHO, 2005;Sarker et al., 2018). A study revealed that 15.18% of S. aureus stains isolated from street foods were resistant to methicillin. All isolated bacterial colonies were resistant to Penicillin G. The bacterial colonies resistant to methicillin were also resistant to kanamycin, gentamycin, tobramycin and erythromycin (Mamun et al., 2013;Islam et al., 2018). Moreover, the Salmonella species isolated from street foods vended in Khulna were sensitive to most of the drug tested. In a similar study, the isolated Shigella species showed multiple drug resistance patterns against ampicillin, trimethoprim-sulphamethoxazole, chloramphenicol, streptomycin and tetracycline. Since the popularity of street foods is increasing in the towns/cities in particular and in the country in general, this study was intended to assess the microbiological quality and antibiogram status of bacterial isolates from ready-to-eat foods sold by street venders to ensure health status of the consumers.

Sample collection
A total of 20 food samples were randomly chosen and collected from street vendors in the area around Khulna city. These samples were collected in different sealed poly bags to prevent their contact with any other source that can contaminate the samples. Food samples included Chotpoti, Chanachur, Amra and Jolpai. Approximately 200g of each food sample was collected using the vendors serving utensils, take parcel and placed into sterile plastic bags. All the collected samples were kept on an icebox during transportation to the laboratory and stored at 4°C until testing. They were analyzed within 24 hours of sampling.

Preparation of sample
Adequate amount of different street food (Chotpoti, Chanachur, Amra and Jolpai) samples were uniformly homogenized in mortar and pastel using a sterile diluent as per recommendation of (Balamurugan et al., 2013). A homogenized suspension was made with the help of mortar and pastel. A quantity of 10 gm homogenate sample of each different street was taken aseptically with a sterile spoon and transferred carefully into a sterile pastle containing 90 ml of PBS. Thus 1:10 dilution of the samples was obtained.

Enumeration of total viable count (TVC)
50µl of each fivefold dilution was transferred and spread onto Plate Count Agar using a micropipette for each dilution for the determination of total bacterial count. The diluted samples were spread as quickly as possible on the surface of the plate. The plates were kept in an incubator at 37°C for 24 hrs. After incubation, plates exhibiting 30-300 colonies were counted. The average number of colonies in particular dilution was multiplied by the dilution factor to obtain the total viable count. The total viable count was calculated according to ISO (1995). The results of the total bacterial count were expressed as the number of colony forming units (CFU) per ml of food samples.

Isolation of associated bacteria
Bacteriological examination was carried out using standard method for aerobic bacteria (Brown, 2005). Each sample of Chotpoti, Chanachur, Amra and Jolpai samples were inoculated separately in nutrient broth (NB) to promote growth of bacteria. Each group of these media were incubated at 37°C for overnight. The colonies on primary cultures were repeatedly subcultured by streak plate method (Cheesbrough, 1985) until the pure culture with homogenous colonies were obtained. Media such as Nutient agar, MacConkey agar, Eosin Methylene Blue agar, Salmonella Shigella (SS) agar, and Manitol Salt Agar (MSA) were used for sub-cultures and incubated at 37°C for 24 hours for growth.

Identification of associated bacteria
The cultural examination of street food (Chotpoti, Chanachur, Amra and Jolpai) samples for bacteriological study was done according to the standard method International Commission on Microbiological Specifications for Foods (ICMSF, 1985). Identification of bacteria was performed on the basis of colony morphology Gram's staining reaction and biochemical test. Biochemical tests, such as sugar fermentation, coagulase, catalase, MR, VP, and indole tests, were performed as per the standard methods (Cheesbrough, 1985).

Results of bacteriological investigation
A total of 20 different street food (Jolpai, Chotpoti, Chanachur and Amra) samples were collected from different places in Khulna for this study. Among 20 different street food (Jolpai, Chotpoti, Chanachur and Amra) samples, Shigella spp., Staphylococcus spp., Klebsiella spp., Salmonella spp. and Escherichia coli were found.

Results of isolation of bacteria
Cultural characteristics of each type of bacteria isolated from different street food were studied for the determination of size, shape and colony characteristics in various bacteriological media. The staining property of primary culture of each of the different street food samples indicated the presence of more than one type of bacteria in the same smear. The pure cultures of the organism from each mixed culture were obtained by repeated streak plate method using different simple and selective solid media for study. The cultural characteristics of Klebsiella spp., Staphylococcus spp., Shigella spp., Salmonella spp. and E. coli exhibited on the media are presented in Table 3.

Results of staining characteristics of the isolated bacteria
The staining characteristics of the isolated organisms were determined according to Gram's staining technique. Morphological and staining characteristics of bacteria recorded from Jolpai, Chotpoti, Chanachur and Amra samples by Gram's staining are presented in Table 4.

Conclusions
In general, this study demonstrated that street vended foods which are sold on the streets of studied areas were considerably contaminated. The foodborne bacteria and antibiotic resistance isolates detected in this study are also evident that street foods might pose a major problem for public health. Lack of training (orientation) on the proper handling and processing of food, poor personal hygiene of venders and unhygienic surroundings could be possible factors for observed problems in that locality. Therefore, education for vendors on food safety and hygienic practices is essential to reduce contamination rate. In addition, regular inspection on food vending practices and safety of street foods is required to improve the health standards of consumers.