Tomato (Lycopersicon esculentum) ranks as the second most extensively cultivated vegetable crop globally. While it is commonly consumed fresh, its highly perishable nature necessitates widespread industrial processing into products such as tomato juice, purée, and paste (Ray et al., 2016). A substantial proportion of the global tomato harvest is industrially transformed into tomato paste, which serves as a fundamental ingredient in a wide array of culinary products, particularly soups, sauces, and ketchup formulations (Gould, 2013). More than 80% of tomatoes farmed commercially are used to make processed foods like juice, soup, and ketchup (Viuda et al., 2014; Happy et al., 2018). 

Fast food items are getting popularity due to a variety of flavors and categories. The consumption of fast food is now almost inseparable from the use of various sauces, which have become integral to enhancing flavor and appeal. Individuals across all age groups from school and university students to working professionals demonstrate a growing preference for fast foods accompanied by diverse sauces, reflecting their escalating popularity and widespread acceptance (Ahmed et al., 2019). Lycopene, potassium, iron, folate, and antioxidants are among the minerals and phytochemicals that tomatoes provide in reasonable amounts (Bhowmik et al., 2012). Other antioxidants including beta-carotene and phenolic substances like flavonoids, hydroxycinnamic acid, chlorogenic, homovanillic acid, and ferulic acid are also present in tomatoes in addition to lycopene and vitamin C. (Borguini et al., 2009). Tomatoes can be a vital component of a nutritious diet and can be eaten either fresh or cooked without losing any of its nutritional content (Bhowmik et al., 2012). Many flavors of fast food are created by varying and cross-combining ingredients. Different restaurants prepare signature spreads to attract customers, but this can backfire if they are not prepared with high-quality raw materials and proper hygienic conditions are not maintained (Tasnia Ahmed and Md. Aftab Uddin, 2020). Consumption of street-side foods is dramatically increasing day by day because of their easy access, attraction, flavor, and appearance. Despite the widespread popularity of street foods across various regions of the world primarily driven by their affordability there remain significant concerns regarding their hygienic standards and overall food safety (Alimi, 2016; Rahman et al., 2019).

In Bangladesh, traditional street-side delicacies such as hog plum pickles, tomato pickles, and carrot pickles enjoy immense popularity (Faruque et al., 2010). A study reported that approximately 62–78% of roadside vendors across the country sell such fermented or preserved items, with 58–66% of these establishments operating in close proximity to open drains, sewer lines, manholes, or garbage disposal sites. Additionally, around 94% of these vendors rely on municipal tap water for serving drinking water, a source frequently identified as microbiologically contaminated (Faruque et al., 2010; Parveen et al., 2008). In many instances, this water is stored in uncapped plastic containers, leaving it exposed to the environment and thus increasing the risk of secondary contamination (Khairuzzaman et al., 2014).

The inadequate hygienic practices associated with the preparation and handling of street-vended foods (SVFs) significantly elevate the potential for foodborne illness outbreaks on a global scale. A critical contributing factor is the limited awareness among street food vendors regarding the etiological agents of foodborne diseases (Appendix E, International Microbiological Criteria, 2003). Escherichia coli (E. coli), a common enteric bacterium found in the intestinal tracts of humans and other warm-blooded animals, exemplify such pathogens. Newborns, initially possessing a sterile gastrointestinal tract, are typically colonized by E. coli within the first 48 hours of life (Abdul Karim et al., 2021).

The food and the hygienic environment in which it is being served lay an impact on the overall food quality. It also emphasizes whether the food is safe for intake or not. All of the aforementioned circumstances are likely to result in food cross-contamination. Sewage and contaminated water have been linked to Salmonella spp., Shigella spp., Campylobacter spp., and E. coli contamination of food (Odonkor and Addo, 2018).  Unsanitary practices such as peeling, slicing, handling, trimming, packaging, and so on can lead to bacterial contamination of foodstuffs (Barro et al., 2006).  Some Bangladeshi Street vendors appear to disregard the importance of personal hygiene when handling food (Faruque et al., 2010). Furthermore, the handmade street foods in Bangladesh are mostly kept at temperatures ranging from 0 to 50ºC, which promotes the growth of a variety of mesophilic pathogenic bacteria in the foods, including E. coli, Staphylococcus aureus, Bacillus spp., Klebsiella spp., Pseudomonasspp., and S. typhi (Barro et al., 2006; Mensah et al., 2002). It is estimated that approximately 30 million cases of food-borne illness occur in Bangladesh each year (Khairuzzaman et al., 2014). In previous several related research was conducted throughout the world but northern part of Bangladesh very limited number of research was conducted about processed tomato sauces that usually used in roadsite fast food restaurants. Most of the person who working in local hotel and restaurant have no proper personal hygiene practices during food servings. Due to unhygienic conditions foodborne bacteria easily transmitted into consumers health through food consumption and create foodborne illness. From the above statement this research is highly significant for public health and very challenging for prevent foodborne illness in human beings. Therefore the main objectives of this study was to identify pathogenic bacteria isolated from different process tomato sauces and their antimicrobial sensitivity tests. 

Study area selection 

In this study, 60 samples of locally produced tomato sauce, used by street food vendors, were collected from various vendors in Dinajpur town, Bangladesh. The tomato sauces samples were aseptically transferred to the bacteriology laboratory at Hajee Mohammad Danesh Science and Technology University for bacteriological analysis. The research was conducted from January to June 2022. 

Sample preparation and processing

All the samples were taken aseptically with transparent zipper lock poly (thickness 30-100mic; size 175mm*100mm) and transferred bacteriology laboratory for microbiological analysis. 10 gm of tomato sauces mixed with 90 ml distilled water. After proper mixing the samples, serially diluted with 10 fold serial dilution and then transferred 0.1 ml samples into nutrients broth for primary culture. Then inoculated test tube incubated at 37°C for 24 hours in BioBase incubator, China. 

Fig. 1: Tomato sauces sample collection from different street food shop, Dinajpur.

Isolation and identification of isolates

Bacterial isolates were initially transferred from nutrient broth to nutrient agar and incubated at 37° C for 24 hours (BioBase, China). After overnight incubation colonies were transferred into  MacConkey agar for bacterial differentiations, and finally subculture on Eosin Methylene Blue agar to isolate pure bacterial colonies from the tomato sauce. Then we prepare gram staining slide iunder microscope for gram reaction determination. For furtur confirmation a group of standard biochemical tests, including MR-VP, Indole, MIU, Catalase, Oxidase, TSI, and Citrate utilization tests were performed according to Azam et al. (2023). All microbiological media used in this study were purchased from Hi Media Private Ltd., India.

Molecular detection of E. coli

The double boiling centrifugation method for DNA extraction was performed as described by De Medici et al. (2003). A single E. coli colony from EMB agar was suspended in 200 µL of DNAse/RNAse-free distilled water. The suspension was incubated at 100°C for 15 minutes, chilled on ice for 5 minutes, and centrifuged at 13,500 rpm for 10 minutes. The supernatant was transferred to a new tube, subjected to a second 15-minute incubation at 100°C, followed by 5 minutes on ice. A 5 µL aliquot of the resulting supernatant served as the DNA template for PCR.

For amplification of the E. coli 16S rRNA gene, primers designed by Tsen et al. (1998) - 16E1 (F): 5′-GGG AGT AAA GTT AAT ACC TTT GCT C-3′ and 16E2 (R): 5′-TTC CCG AAG GCA CAT TCT-3′ - were employed. Additionally, Gautam et al. (2012) used an alternative reverse primer, 16E3 (R): 5′-TTC CCG AAG GCA CCA ATC-3′. PCR was conducted in a 25 µL reaction containing 12.5 µL of 2x GoTaq Green Master Mix (Promega, USA), 2 µL of DNA (diluted to 50 ng/µL), 0.2 µL Taq DNA polymerase, 0.5 µL each of forward and reverse primers, and 9.5 µL of molecular-grade water. PCR cycling conditions are detailed in Table 1. The purity of PCR products was evaluated using a Thermo Scientific NanoDrop 2000 spectrophotometer by measuring the A260/A280 absorbance ratio. Post-electrophoresis, gels were transferred from the electrophoresis chamber to a UV transilluminator (WUV-L50, Korea) for preliminary visualization of DNA bands. Subsequent analysis and image documentation were performed using a high -performance gel documentation system (UVD1-254). PCR products were resolved on a 2% (w/v) agarose gel containing 0.5 µg/mL ethidium bromide, run at 70 - 100 V and 500 mA for 30 - 70 minutes, as described by Aklilu et al. (2016). A 100 bp DNA ladder (Thermo Scientific, USA) was used as the molecular size marker. Gel images were captured using a high-resolution camera equipped with Vtech software and a TV zoom lens (Japan).

Table 1: PCR Conditions for E. coli.

Antibiotic Susceptibility Test (AST)

According to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2017), the agar disc diffusion method was employed to assess the antibiotic sensitivity patterns of the isolates on Mueller-Hinton agar. A total of 10 commercially available antibiotic discs were used, including Ampicillin (25 µg), Ciprofloxacin (25 µg), Ceftriaxone (5 µg), Enrofloxacin (30 µg), Cefixime (5 µg), Azithromycin (5 µg), Levofloxacin (30 µg), Tetracycline (30 µg), Amoxicillin (30 µg), and Doxycycline (30 µg). A pure bacterial colony (0.1) was inoculated onto a Mueller-Hinton agar plate, and the antibiotic discs were placed on the surface of the plate. 

The plates were then incubated overnight at 37°C in a standard incubator (BIOBASE, China). After incubation, the zones of inhibition were measured in millimeters according to CLSI (2017) guidelines. All tests were conducted in triplicate to ensure the accuracy of the results. All antibiotics were purchased from HI Media Private ltd., India.

Multidrug and Multiple Antibiotic Resistance (MAR) calculation 

An isolate was considered multidrug-resistant (MDR) if it showed resistance to two or more classes of antibiotics on Mueller-Hinton agar plates. According to (Ezekiel et al., 2011), the multidrug resistance pattern was determined. The MAR Index (M) is calculated using the formula: 

M =a /b

Where: "a" = Number of antibiotics to which the isolate is resistant, and "b" = Total number of antibiotics tested.

Statistical Analysis

All data were initially entered into a Microsoft Excel spreadsheet and subsequently analyzed using SPSS version 25. Statistical comparisons between group means were conducted using one-way analysis of variance (ANOVA), followed by appropriate post hoc tests to assess pairwise differences. The zone of inhibition values are expressed as mean ± standard deviation, and the standard error of the mean, with statistical significance determined at a threshold of P < 0.05. P- value of descriptive analysis was calculated by using R Studio version 2024.12.1+563.

Sauces are very popular nutritive food additives which are especially consumed with fast foods. Fast food is becoming more popular as a result of the variety of flavors and categories available and eating fast food without sauces is unthinkable (Bhowmik et al., 2012). A tomato sauce production process that does not meet the standard may result in microbial contamination of the tomato sauce and bottles. Tightly closed, tomato sauce can be protected from outside influences such as rot causing microbes, but if left open, it increases the risk of ketchup contamination by microbes such as bacteria that could endanger or threaten human health, (Sugiyono et al., 2013). The present study provides valuable insight into the incidence of E. coli isolated from locally produced tomato sauce sold by street food vendors. A total of 60 samples were collected and subjected to various bacteriological and biochemical examinations to detect the presence of E. coli. The findings confirmed the presence of E. coli in 73% (44/60) of the samples, while 27% (16/60) were free from E. coli contamination.

These results are consistent with the findings of Hossain et al. (2019) who reported similar contamination levels in a study conducted in Jashore, Bangladesh. In that study, 30 samples of plum sauce and tomato sauce were analyzed using the dilution plate technique and selective media for microbial identification. All samples contained viable Enterobacteriaceae, with 80% and 83.33% of the samples contaminated with Salmonella spp. and E. coli, respectively. The total viable bacterial counts ranged from 1.2 × 10³ to 4.2 × 10⁹ CFU/g. E. coli counts varied from 0 to 7.0 × 10⁵ CFU/g, and Enterobacteriaceae counts ranged from 30 to 2.0 × 10⁷ CFU/g. Although higher contamination levels were found in plum sauce samples compared to tomato sauce, the difference was not statistically significant (p > 0.05).

The E. coli isolates from the current study were further subjected to antibiotic susceptibility testing. The antibiogram revealed that all E. coli isolates exhibited high sensitivity to ciprofloxacin, ceftriaxone, enrofloxacin, azithromycin, levofloxacin, tetracycline, and doxycycline, while showing resistance to amoxicillin, ampicillin, and cefixime.

The detection of E. coli in locally produced tomato sauce poses a significant public health risk. Therefore, it is imperative to implement strict hygienic guidelines for the production, handling, and distribution of such food products. Public awareness should also be raised regarding the potential health hazards associated with consuming contaminated tomato sauce from street vendors, to reduce the risk of foodborne illnesses.

Tomato sauce is a popular condiment made from tomatoes, vinegar, sugar, salt, and various spices and preservatives. It enhances the flavor of fast food items. Currently, almost all local fast food outlets serve tomato sauce to enhance taste. Most local vendors provide sauce that is not prepared with proper hygiene. In particular, sauces made in unhygienic environments carriers of harmful bacteria like E. coli which can causes diarrhoea in consumer.  The study was undertaken to isolate E. coli and perform a comparative antibiogram analysis of strains derived from locally produced tomato sauce. Antibiotic resistance has emerged as a critical global challenge, as it significantly complicates and prolongs the therapeutic management of infectious diseases like diarrhea, vomiting, and stomach pain. Beside antibiotic resistance Ciprofloxacin and levofloxacin can be used as potential sensitive antibiotics against diarrhoea causing E. coli  that significantly found in locally prouced tomato sauces.

Conceptualization, Investigation, Writing Reviews: M.R.A.; M.F.: Original draft writing: M.R.A.; S.M.; M.F.: Data Collection and analysis: M.R.A.; M.F.A.; M.S.H.M.: Laboratory experiment: M.R.A.; M.T.I.S.: Critical Reviews: M.K.H.; M.F.: Editing: M.T.I.S.; M.F.: Reference preparation: M.S.H.M.; Supervision: M. F.; and M.K.H.: Before final submission, all assigned authors checked the full manuscript and approved for publication. 

The authors have no conflicts of interest. 

The research was not directly involved with animal and human thats why no ethical clearance was needed. According to Departmental research guidelines we conducted our research work in Bacteriology laboratory at Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh. 

Authors give special thanks to the Department of Microbiology, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh for their Laboratory facilities.