Street food is ready-to-eat food in a street or other public place, such as a market or fair, by a hawker or vender, often from a portable food booth  food cart or food truck. The street foods are the well appreciated by consumers, due to its taste, low cost, nutritional value and availability for immediate consumption  . Contamination might also occur due to poor food handling practices of the vendors as most vendors lack awareness and knowledge of handling of food safety and have not been formally trained in food safety  . Small fried fish, a street food in ancient Greece  although Theophrastus held the custom of street food in low regard.
Over the years, many food-borne diseases have been reported due to contaminated non-homemade food consumption. Street food was widely utilized by poor urban residents of ancient Rome whose tenement homes did not have ovens or hearths, with chickpea soup being one of the common meals along with bread and grain paste  . In ancient China, where street foods generally catered to the poor, wealthy residents would send servants to buy street foods and bring meals back for their masters to eat in their homes  . As of 1707, after previous restriction that had limited their operating hours, street food vendors had been banned in New York City  . Most of the members of Enterobacteriaceae cause gastrointestinal problems in human body  . In the 1800s street food vendors in Transylvania sold ginger bread-nuts, cream mixed with corn and bacon and other meat fried on tops of ceramic vessels with hot coals inside.
Bangladesh is populated with many vendors of street foods of different kinds. Like all developing countries, street food preparation and selling in Bangladesh provides a regular source of income for millions of men and women with limited education or skills. Almost all of the bacteria are pathogenic to human at various degrees and infections caused by some bacteria are even hard to treat with increase the rate of antibiotic resistance in the community  . The prevalence and growth of all pathogens on the raw foods especially vegetables, salads, fruits and sprouts, are used as ingredients of chutneys   . Especially the rickshaw puller, laborers eat street foods just to satisfy their hunger. Rickshaw puller, laborers, informal sectors (43%), white-coller workers (19%) student’s children (12%), others (26%)  . There are 128 varieties of street foods found in Bangladesh. Among them Chotpoti, Belpuri, Samucha, Jhalmuri, Daalpuri, Lassi, Pakura, Halim are most popular  .
Presence of antibiotic resistant bacteria in street-foods has become a global health concern. Street food vending has become an important public health issue and a great concern to everybody. Microbial food borne illness is the major health problem associated with street foods  . Food handlers can also become carriers of the pathogenic food microorganisms such as Salmonella spp  . Some of the phenolic compounds present in various foods have antimicrobial activity against gram negative bacteria  . Street food importance has consequences such as its association to epidemic and disease outbreak in case of the microbiological quality failure  . The objectives of this study were microbiological analysis of Chotpoti, to find out the presence of enteric bacteria and to describe the characteristics and practices with regard to street food among the people.
2. Materials and Methods
2.1. Study and Sample Collection Site
The study was conducted during the period from June to December 2014, in the Department of Microbiology, Gono Bishwabidyalay, Savar, Dhaka. Chotpoti samples were collected from different street vendors at Ghorapir Mazar stand (23˚55'31.7"N 90˚14'41.4"E), Pollybidut stand (23˚54'49.5"N 90˚15'29.0"E) and Nabinagar bus stand (23˚54'49.5"N 90˚15'29.0"E). All the samples of this study were collected aseptically using sterile instruments and transferred carefully to appropriate containers. The samples were carefully handled and kept in box. Due aseptic care was taken during transportation and the samples were kept in sterile container until they are prepared for bacteriological analysis.
2.2. Media and Reagents
The media used for bacteriological analysis were MacConkey agar, Eosin-Methylene-Blue (EMB) agar, Salmonella-Shigella (SS) agar, Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar, Muller Hinton agar, Nutrient broth (NB) and Peptone broth. The reagents used during the study were Lacto phenol-cotton-blue solution, Crystal violet, Gram’s iodine, Safranin, 95% Ethyl alcohol,3% hydrogen peroxide, Kovac’s & indole reagent, Burritt’s reagent A and B, normal physiological saline solution. Muller Hinton Agar plates, Phosphate buffered saline (PBS) solution, normal saline and distilled water, etc.
2.3. Preparation of Sample
Portions of Chotpoti samples were uniformly homogenized in mortar and pastel using sterile diluents as per recommendation of ISO. A quantity of 10 gm homogenized sample of each Chotpoti was taken aseptically with a sterile spoon and transferred carefully into a sterile pestle containing 90 ml of PBS. Later on using whirly mixture machine different serial dilutions ranging from 10-2 to 10-4 were prepared according to the standard method.
2.4. Isolation and Identification of Bacteria from Chotpoti
Homogenized samples were enriched into alkaline peptone water by overnight incubation at 37˚C. Overnight enriched culture was streaked and spreaded onto Salmonella-Shigella (SS) agar, Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar and incubated at 37˚C for 24 hrs. The cultural examination of Chotpoti samples for bacteriological analysis was done according to the standard method. Identification of bacteria was performed on the basis of colony morphology; Gram’s staining reaction and biochemical test.
2.5. Antibiotic Discs and Media Preparation
Commercially available antibiotic disc has been used for the test to determine the drug sensitivity pattern. The commercially media were prepared according to the direction of the manufactures and the non-commercial media were prepared in the laboratory (Table 1).
2.6. Enumeration of Total Viable Count (TVC)
0.1 ml of each ten-fold dilution was transferred and spread on duplicate onto NA, EMB and MacConkey 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 with sterile glass spreader. The plates were kept in an incubator at 37˚C for 24 hours. The results of the total bacterial count were expressed as the number of colony forming units (CFU) per gram of food samples.
2.7. Colony Characteristics
Colony characteristics such as shape, size, surface texture, edge and elevation, color and opacity developed on various selective media after 24 hrs of incubation at 37˚C were recorded.
2.8. Gram’s Staining
Gram’s staining of the pure culture was performed according to method described
Table 1. Antimicrobial agents with their disc concentration.
by Aneja et al. (2003). Briefly, a single colony was picked up with a bacteriological loop, smeared on separate glass slide and fixed by gentle heating. Crystal violet was then applied on each smear to stain for two minutes and then washed with running tap water. Few drops of Gram’s iodine were then added for few seconds. After washing with water, Safranin was added as counter stain and allowed to stain for 2 minutes. The slides were then washed with water, blotted and dried in air and then examined by a light microscope (100×).
2.9. Biochemical Tests
Biochemical tests were done in accordance to standard procedure  . Traditional media culturing and biochemical characterization of Enterobacteriaceae are still an authentic widely used methods for isolation and identification of bacteria from many sources including consumable foods  .
2.9.1. Catalase Test
This test is used to different those bacteria that produce the enzyme catalase. To perform the test an amount of 2 - 3 ml of 3% hydrogen peroxide solution was poured into a test tube. If the organisms are catalase producer, bubbles of oxygen are released.
2.9.2. Sugar Fermentation Test
The sugar fermentation test was performed by inoculating a loop full of NB culture of the organisms into each tube containing three basic sugars (e.g. Dextrose) separately and incubated for 24 hours at 37˚C. Acid production was indicated by the color change from reddish to yellow in the medium.
2.9.3. Indole Test
Five milliliter of peptone water was inoculated with the 5 ml of bacterial culture and incubated at 37˚C for 48 hours. Kovac’s reagent (0.5 ml) was added, shaked well and examined after 1 minute. A red color in the reagent layer indicates indole.
2.9.4. Voges-Proskauer Test
Five milliliter sterile glucose peptone water was inoculated with the 5 ml of test organisms. It was incubated at 37˚C for 48 hours.
2.9.5. Triple Sugar Iron (TSI)
This tube medium is used to identified Gram negative enteric bacteria based on few biochemical characteristics includes―glucose indicated by yellow butt, lactose fermentation-indicated by yellow slant and gas production indicated by presence of a crack or gas space, etc.
2.9.6. Urease Broth
This tube medium is used to identify Gram negative enteric based on the ability of the organism to produce the enzyme urease within 2 to 4 hours (rapid) or within 24 hours (late) of incubation. If the organism produces urease, this splits urea into ammonium, carbon-di-oxide and water.
2.10. Antibiotic Sensitivity Test
Antimicrobial drug susceptibility against two commonly used antibiotics was performed by disc diffusion method. The procedure of disc diffusion method is the isolated microbial colony was inoculating into Nutrient-Broth and incubated at 37˚C for 24 hours. The plates were inverted and incubated at 37˚C temperature for overnight. After incubation the diameter of the zone of complete inhibition (including diameter of the discs) was measured in millimeters with a ruler.
3. Result and Discussion
3.1. Total Viable Count (TVC) of Chotpoti
The TVC of Chotpoti samples collected from different vendors are presented in Table 2. Thirty-fold dilution was done to obtain single bacterial colony. In the current study, the highest TVC of Chotpoti was found in Nabinagar bus stand and the lowest TVC recorded in Pollybidut bus stand vendor (3 × 105 CFU/g).
3.2. Total Coliform Count (TCC) of Chotpoti
The TCC of Chotpoti samples collected from different vendors are presented in Table 3. Twenty-fold dilution was done to obtain single bacterial colony. The highest TCC of Chotpoti was found in Nabinagar bus stand vendor (3 × 106 CFU/g) and the lowest TCC recorded at Pollybidut bus stand vendor (TNTC). The contamination might take place during processing, preparation and handling of the Chotpoti (Figure 1 & Figure 2).
3.3. Isolation and Identification of Bacteria from Chotpoti Sample
The isolation of coliform bacteria from Chotpoti sample indicates fecal contamination. Presence of coliforms in the present study might be due to poor quality
Table 2. Total viable count of Chotpoti sold by vendors at Savar area.
Table 3. Total coliform count of Chotpoti sold by vendors at Savar area.
EMB = Eosin Methylene Blue.
Figure 1. Total coliform count on EMB agar plate.
Figure 2. Bacterial colony on MacConkey agar plate.
of water used for washing of fruits, vegetables and utensils, inadequate storage of these at ambient temperatures in unhygienic places, maintenance of premises and personal hygienic of vendors. Microbiological studies carried out on street-food vending in several developing countries have reported high bacteria counts in food  . Identification of bacteria was performed by determining cultural characteristics, staining reactions and different biochemical properties. In the current study, four different genera of bacteria such as Escherichia coli, Klebsiella spp., Salmonella spp. and Shigella spp. were identified (Table 4).
3.4. Cultural Characteristics
The cultural characteristics of isolated bacteria exhibited on the selective media are presented in Table 5. Street foods are usually doing not receive any treatment as a result they are prone to get contaminated with food borne and antibiotic resistant bacteria that may cause public health hazard.
3.5. Results of Antibiotic Sensitivity Tests
A total of 08 isolates of 04 genera such as Escherichia coli, spp Klebsiella spp., Salmonella and Shigella spp. were subjected to antibiotic sensitivity assay. The results of antibiotic sensitivity assay are presented in Table 6.
In this antibiogram sensitivity test some specific commonly used antibiotics
Table 4. Summary of isolated bacteria from Chotpoti sold at different sources.
Table 5. Cultural characteristics of the bacterial isolates of Chotpoti.
Table 6. Resistant and sensitive patterns of the isolates of Chotpoti samples.
Interpretation: R = resistant, S = sensitive.
are used against the specific microorganism. In this study, gram negative bacteria were found multidrug resistant. Antibiotic against which bacterial isolates of Chotpoti were found resistant were Penicillin G, Tetracycline and Vancomycin.
3.6. Gram’s Staining Results
In this study, microbiological methods were used to identify bacteria isolated from Chotpoti samples. The results of cultural characteristics, Gram’s staining, used to identify Escherichia coli, Klebsiella spp., Salmonella spp. and Shigella spp. were identified.
3.7. Biochemical Test Results
The biochemical identification of E. coli, Klebsiella spp. and Salmonella spp. were done by performing the biochemical test. The biochemical test was done as started on Bargen’s manual of Determination Bacteriology. In many other studies, E. coli was detected frequently in street food samples  . In this project 14 biochemical test has been done. Based on the result the major gram negative pathogenic bacteria have been identified. A total 08 gram negative bacterium was isolated from Chotpoti samples (Table 7).
Street foods are very much popular in the recent days. Improper personal hygiene can facilitate the transmission of the pathogenic bacteria found in environment and on people’s hands via food to humans. Street foods have unique flavor, are easily accessible and cheap  . Hygiene in handling and cooking of street foods is very essential and personal hygiene is also very much important for food safety because human are the largest source of contamination. From this study, it clear that all the samples are microbiologically unacceptable to eat. A study on various types of ready-to-eat-food samples in Alice, South Africa showed bacterial growth in all the food types tested and found 22% of samples contaminated with Listeria spp. 18% with Enterobacter spp. 12% with Aeromonas hydrophila, 8% with Klebsiella oxytoca, 6.3% with Proteus mirabilis, 3.2% with Staphylococcus aureus and 2.4% with Pseudomonas luteola  . Strict public health regulations should be established to control the situation. The maintenance of these street vended foods should be monitored cautiously. Insects and rodents carry pathogens from garbage, wastewater, and overloaded drain during
Table 7. Different biochemical test results with their respective microorganisms.
MR: Methyl red; VP: Voges-Proskauer; AG: Acid and gas; A: Acid; (+) = positive; (−) = negative. Comments: Isolate number: 1 (A), 2 (B) = Escherichia coli; Isolate number: 1 (B), 2 (C), 2 (D) = Klebsiella spp.; Isolate number: 2 (A), 3 (A) = Salmonella spp.; Isolate number: 2 (E) = Shigella spp.
their nutrient intake  . The government should take necessary steps to provide regular training and to create consciousness on food management and individual hygiene among street food vendors as well as consumers. Data of this study suggest that Chotpoti sold by street vendor contaminated with multidrug resistant food borne bacteria might pose public health hazard. For continuation of the present study the following research theme may be advocated to be conducted in future; pathogenic characterization of bacteria and molecular characterization of isolates by PCR analysis.