Introduction
Street foods consist of ready-to-eat items and drinks prepared and sold by vendors in urban and public settings.1 They are a vital part of modern eating habits and a significant contributor to the informal urban sector in developing countries. Approximately 2.5 billion people eat street food every day globally2 and India is no exception with an estimated 20 lakh vendors serving street foods.3
However, there are many concerns about its safety. People consuming street-vended foods are known to experience foodborne diseases like diarrhea, typhoid fever, and food poisoning4 and the majority of them go unreported. Global estimates of foodborne diseases revealed that The African and South-East Asia regions are the most affected by foodborne diseases.5 A recent report indicated that foodborne disease cases in India are projected to increase from 100 million to between 150 and 177 million by 2030. This implies that, on average, one in nine people is expected to fall ill from foodborne illnesses by 2030.6
Diarrheal disease agents were the leading cause of foodborne disease burden, and non-typhoidal Salmonella enterica is an important burden globally. Other main organisms include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli and Vibrio cholerae, and Campylobacter spp. These disease-causing agents have been reported in street foods.7, 8, 9
Street foods in recent times are becoming a new food vehicle for emerging foodborne diseases. Emerging, re-emerging, or drug-resistant pathogens have recently appeared in the population. While these pathogens have been widespread for many years, they have only recently been identified due to advancements in knowledge and methods for detecting and analyzing the disease agents.10 Emerging foodborne pathogens can include both Gram-negative and Gram-positive bacteria.11 Reports have shown emerging strains of bacteria in street foods as well.12, 13, 14
Microbial contamination in street-vended foods has become a pressing concern. Thorough studies of the microorganisms found in these foods are crucial for assessing the toxicity of foodborne pathogens. At present, there is limited data on the microbial contamination of street foods in developing countries like India. This study aimed to identify high-risk and emerging foodborne pathogens in commonly consumed street foods in Hyderabad, a city in South India.
Materials and Methods
Study area
Hyderabad, the capital of the state of Telangana, has a population of approximately 6.9 million, with a metropolitan population of around 7.75 million. This makes it the fourth most populous city and the sixth most populous urban agglomeration in India. The city features a significant informal economy, with 40,000 to 50,000 street vendors and a growing number. In this study, 300 street food samples were collected from five zones of Hyderabad—east, west, north, central, and south—encompassing 18 circles and 150 wards under the Greater Hyderabad Municipal Corporation (GHMC) for microbiological analysis.
Study design, sampling technique, Inclusion criteria
It was a cross-sectional study. A stratified random sampling technique was applied and samples were stratified into zones and randomly collected from each zone. Street food vendors who are typically on streets without basic infrastructure (Cement roof, Furniture, Drinking water supply, etc.)
Sample collection and processing
A total of 300 street food samples were collected, including 150 samples of Chinese fast foods (comprising 50 chicken noodles, 50 chicken fried rice, and 50 chicken - 65) and 150 samples of Bhelpuri (a mix of puffed rice, vegetables, and sauces). The samples were transported in sterile polythene bags, kept at 4-10°C, and analyzed within 2-4 hours of arrival at the laboratory. Each sample, weighing 25 grams, was added to 225 ml of sterile buffered peptone water. The resulting dilutions were then inoculated onto appropriate media for further analysis.
Pathogen and indicator organism analysis
The identification and enumeration of foodborne pathogens and hygiene indicators—E. coli, Fecal coliforms, S. aureus, Salmonella spp., Shigella spp., and Bacillus cereus—were conducted following the standard procedures outlined in the Bacteriological Analytical Manual (USFDA-BAM).
E. coli: Identified by spreading 0.1 mL of a diluted sample onto sterile MacConkey Agar (Hi Media Laboratories Pvt. Ltd.) and incubating at 35°C for 24 hours. Pink colonies were considered presumptive E. coli. Further confirmation was done by streaking colonies onto EMB agar for metallic sheen and performing biochemical carbohydrate fermentation tests.
S. aureus: Detected by spreading 0.1 mL of a diluted sample onto sterile Baird Parker Agar (Hi Media Laboratories Pvt. Ltd.) and incubating at 35°C for 24 hours. Golden yellow colonies indicated presumptive S. aureus. The presence of coagulase was tested to confirm coagulase-positive strains.
B. cereus: Isolated on Bacillus Cereus Agar (Hi Media Laboratories Pvt. Ltd.) supplemented with egg yolk and Polymyxin B. Diluted samples (0.1 mL) were spread on plates and incubated at 30°C for 48 hours. Blue colonies were presumptive B. cereus and were confirmed through Gram staining and nitrate reduction tests.
Salmonella spp.: Detected by spreading a 1:10 dilution of the food samples onto Salmonella-Shigella Agar (Hi Media Laboratories Pvt. Ltd.) and incubating at 37°C for 24 hours. Jet black colonies with translucent edges were indicative of Salmonella spp., confirmed by a Latex agglutination test.
Shigella spp.: Identified by spreading a 1:10 dilution of food samples onto Salmonella-Shigella Agar (Hi Media Laboratories Pvt. Ltd.) and incubating at 37°C for 24 hours. Transparent colonies were considered presumptive Shigella and confirmed by biochemical tests.
Enterotoxin detection of S. aureus
The S. aureus cultures were tested for toxin production This was done by an ELISA kit provided by R-Biopharma AG, Darmstadt, Germany.
Pure cultures have been prepared and stored on slant agar tubes and glycerol stocks. Selected isolates have been subjected to further characterization.
DNA isolation & 16s rRNA sequencing
DNA isolation was done using a column-based DNA extraction kit supplied by Bioserve Biotechnologies Pvt. Limited, Hyderabad. Quantification of DNA was done using the NanoDrop spectrophotometer. Sequencing was performed with 5 different primers designed in the conserved regions on 16s rRNA i.e., 16SEQ2R, 16SEQ3F, INS16SREV, 16SEQRF, 16SEQ4R.
Antibiotic sensitivity of isolated foodborne pathogens
Antimicrobial sensitivity was assessed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (Hi Media Pvt. Ltd.). The study employed eight of the most commonly prescribed antibiotics by physicians for this evaluation.
Ethical approval: This study was granted ethical clearance by the Institutional Ethical Committee at the National Institute of Nutrition, Hyderabad (Registration No: ECR/35/Inst/AP/2013)/ Participation was voluntary, with all street vendors being fully informed about the study's objectives and procedures. To ensure confidentiality, participants were identified by code numbers instead of their names.
Results
Prevalence of foodborne pathogens and hygiene indicator organisms
Bhel puri samples showed a high prevalence of indicator organisms, with E. coli detected in 60.6% of the samples and fecal coliforms in 67.3%. Bacillus cereus which is another most common pathogen in cereals and cereal products was detected in 67.3% of the bhelpuri samples. Enteropathogens such as Shigella and Salmonella were identified in 45.3 and 20% of samples respectively.
Among Chinese fast foods, Bacillus cereus was detected in 90-92% of samples. About 28-40% of samples were observed positive for S. aureus with the highest in Chinese fried rice. Indicator organisms were detected in 16-24% of Chinese foods followed by Shigella spp. Salmonella spp. was detected in only 3 samples, as detailed in Table 1.
Zone (East, West, North, South, and Central) wise comparison of contamination in street food samples of Hyderabad
Bhelpuri- Among all the zones, north and south zone samples have shown high percentages of bacterial contamination. The lowest prevalence (not exceeding 30%) was observed in the samples from the central zone. Bacillus cereus (85%) and Fecal coliforms (92%) were more prevalent (Figure 1). Chinese fast foods- Bacillus cereus was more prevalent (80-99%) in all the samples of Chinese fast foods and followed the same trend in all the five zones of Hyderabad (Figure 2).
Microbial population in high-risk street foods
Bhelpuri samples were contaminated with fecal coliforms (2.59 log) and Bacillus cereus (2.47 log) followed by E. coli (2.27 log) and Salmonella spp. (2.16 log). Chinese fast foods were contaminated with Bacillus cereus (3.65-4.01 log). The minimum and maximum load of bacteria were also presented in Table 1. The minimum log value was two and the maximum value was five log units.
Table 1
Prevalence of foodborne pathogens and hygiene indicator organisms in street foods with the percentage in parenthesis in Hyderabad
Antibiotic sensitivity of isolates from street foods
About 81 isolates have been used to check the antibiotic sensitivity with nine commonly prescribed antibiotics. All the isolates (n=6) of Salmonella spp. were sensitive to the antibiotics except for one isolate which was resistant to Co-Trimoxazole. Among the isolates of Shigella (n=20), 50% were resistant to Co-Trimoxazole, and the majority of 11 isolates to Amoxicillin. All the E. coli isolates have been sensitive to antibiotics, except for Amoxicillin, to which there was a 71% resistance rate. Compared to the other isolates S. aureus was more resistant to the antibiotics with the highest of 85% resistance to Furazonidole, Gentamycin, and Ampicillin. Among the four types of isolates, Salmonella spp. and E. coli showed greater sensitivity to antibiotics, whereas Shigella and S. aureus exhibited higher resistance, as shown in Table 2.
Table 2
Antibiotic sensitivity of foodborne pathogens isolated from street foods with the percentage in parenthesis
Toxic profile of S. aureus
Out of 38 isolates of S. aureus, about 63.18% were coagulase-positive and 36.85% were coagulase-negative (Table 3). Only 1 isolate was detected positive for enterotoxin production and 37 isolates were negative for toxin production.
Molecular characterization of isolates
Of the 300 samples, 11% tested positive for Salmonella spp. and 28.6% for Shigella spp. selected isolates of these pure cultures were processed for molecular characterization. Results have been identified as Salmonella bongori and Shigella sonnei. The dendrograms of these are shown in Figures. 3 and 4.
Discussion
Street foods are highly susceptible to contamination by various foodborne pathogens and indicator organisms. A study conducted in Egypt on street vended meat sandwiches by Lidya et al. 14 had Listeria monocytognes and E. coli O157 H: 7. Another study by Maustafa et al. 15 had L. monocytogenes in 78/576 samples analyzed. A popular street snack Panipuri samples had another emerging foodborne pathogen i.e. Yersinia enterocolitica. 13 Campylobacter jejuni was detected in street-vended grilled chicken samples in Mexico.12, 16
In this study, approximately 67% of the Bhel puri samples were contaminated with Bacillus cereus, likely due to the widespread presence of bacterial spores, especially in dusty roadside environments. The mesophilic spore-formers are introduced into the food through contaminated puffed rice. Previous research by Frazier and Westhoff17 indicated a count of 0.01-0.9 x 10^4 CFU/g of mesophilic bacilli in street-vended foods, attributed to the wheat and rice flour used in preparation. The presence of Bacillus cereus is concerning due to its production of heat-sensitive (diarrheal) and heat-stable (emetic) toxins associated with food poisoning.18 Hanashiro’s findings also revealed that 35% of street food samples were deemed unsuitable for consumption due to high B. cereus loads.19
Staphylococcus aureus and Bacillus cereus are known for their tolerance to a wide range of temperatures and pH levels, which helps them persist even in highly acidic environments. In this study, S. aureus was detected in 26% of samples, while Shigella spp. was found in 45% and Salmonella spp. in 15%. The presence of Shigella spp. and Salmonella spp. may be attributed to inadequate cleaning of vegetables and raw produce used in food preparation.
Chinese fast foods in this study showed a high prevalence of Bacillus cereus, followed by S. aureus, aligning with findings from Sudershan et al.20 The presence of S. aureus could be due to unclean hands or vendors coughing during food preparation. Bacillus cereus contamination is primarily due to spores in raw vegetables, which survive cooking.18 The contamination of street foods with Salmonella spp. is often due to improper washing of salads,21 as observed in Hyderabad, where 41% of vendors used unclean and unpeeled vegetables.22 This practice, coupled with poor personal hygiene, contributes to cross-contamination and reflects poor hygiene standards.23
In terms of antibiotic sensitivity, Salmonella spp. in this study was sensitive to all tested antibiotics, contrasting with studies by Madhuchanda et al.24 and Poonam et al.,25 which reported resistance to Ampicillin, Norfloxacin, Polymyxin, Chloramphenicol, and Amoxicillin. S. aureus showed resistance to Furazolidone, Cotrimoxazole, Gentamicin, and Ampicillin, while Poonam et al. 25 noted resistance to Ampicillin and Cefuroxime. E. coli exhibited resistance to Amoxicillin in this study, consistent with Poonam et al.25 but differing from Madhuchanda et al.,24 who reported resistance to Ampicillin and Chloramphenicol. Most of the isolates from food samples were found to be sensitive to antibiotics, in contrast to isolates from human samples, which exhibited high resistance as reported by Sudershan et al.26 The level of resistance in foodborne isolates was notably lower compared to those from human sources. This difference could be attributed to the extensive use of antibiotics in animal farming, agriculture, and human healthcare, which contributes to the emergence of resistant bacteria in these systems. While humans are less directly exposed to antibiotics than animals, the transmission of antimicrobial-resistant microorganisms at the human-animal interface remains complex.27 Despite limited exposure, bacteria in food samples tend to remain sensitive to antibiotics but may acquire resistance after entering the human system.
The presence of S. aureus alone does not indicate food poisoning; enterotoxin production must be tested to assess toxicity. According to FSSAI standards, foods should not contain coagulase-positive S. aureus. In this study, 63% of isolates were coagulase-positive, similar to Salamandane et al., 28 but only one isolate was positive for enterotoxin production. There are also reports of coagulase-negative 29 S. aureus producing toxins, indicating a need for further research in this area.
In this study, Shigella sonnei was identified as the predominant species. This pathogen had also been reported in earlier research by Mansah et al.,30 who detected S. sonnei in street-vended macaroni, rice, and tomato stew samples. Other species, such as Shigella flexneri, were found in street food samples from studies conducted by Muleta et al.31 and in ethnic street foods from the Himalayas.32 Although S. sonnei has been infrequently found in street food samples, its presence seems to be on the rise.
Historically more common in developed countries, S. sonnei is now emerging in developing regions, including those previously dominated by other Shigella species. The exact reasons behind this shift and the global spread of S. sonnei remain unclear but may be linked to significant environmental pressures. The strain, which originated in Europe, has recently gained a global foothold. Additionally, localized antimicrobial use appears to have exerted strong selective pressure, contributing to the rapid evolution of the S. sonnei population. Consequently, this species is poised to become one of the more dominant and concerning gastrointestinal pathogens soon.33
Another significant organism detected in this study was Salmonella bongori. The Salmonella genus comprises two species: S. enterica and S. bongori, classified based on genetic similarities and biochemical properties.34, 35 While most serovars belong to S. enterica subspecies—such as enterica (about 60%), salamae (20%), diarizonae (13%), arizonae (3.8%), houtenae (2.8%), and indica (0.45%)—only 20 serovars (0.8%) are attributed to S. bongori.
Typically, S. bongori strains are primarily isolated from reptiles and other cold-blooded animals, and human cases involving this species are rare.36 However, in recent years, several strains with the antigenic formula 48, identical to the original isolate, have been identified in hospital laboratories across three major cities in Sicily, Italy. Despite this, limited data exists to trace the specific sources of this serovar. S. bongori is predominantly found in cold-blooded animals, but it can also infect humans. In evolutionary terms, some characteristics of S. bongori suggest that it occupies a middle ground between E. coli and S. enterica. 37
Conclusion
This study highlighted critical insights into the prevalence and load of high-risk foodborne pathogens. It underscores the urgent need for training street food vendors in proper food safety and hygiene practices to reduce the risk of contamination. Research like this contributes to a better understanding of the hazards associated with consuming street foods and provides valuable information to help mitigate those risks.
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