Open Veterinary Journal, (2026), Vol. 16(2): 953-961

Research Article

10.5455/OVJ.2026.v16.i2.17

Molecular detection of the Stx1 and Stx2 genes in Escherichia coli isolated from veal meat sold at retail and butchers’ shops

Hajer Shams Al-Din Abdullah¹, Ashraf Saddik Alias² and Omar Hashim Sheet^3*

¹Department of Environmental Sciences, College of Environmental Sciences, University of Mosul, Mosul, Iraq

²Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine,University of Mosul, Mosul, Iraq

³Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

*Corresponding Author: Omar Hashim Sheet. Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq. Email: omar.sheet [at] uomosul.edu.iq

Submitted: 23/10/2025 Revised: 05/01/2026 Accepted: 19/01/2026 Published: 28/02/2026

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ABSTRACT

Background: The bacterium Escherichia (E.) coli has long been acknowledged as a major causative agent of food-borne illness, with the ability to cause several illnesses and fatalities nationwide. It has the ability to produce Stx1 and Stx2 toxins that cause food poisoning.

Aim: The primary goals of the current study were to isolate and identify E. coli classical methods and confirmed by detecting the uidA gene using a polymerase chain reaction (PCR) assay to identify the Stx1 and Stx2 genes in E. coli isolated.

Methods: Three hundred samples of veal meat sold at retail and various parts of butchers’ shops were collected from each side of Mosul city.

Results: The prevalence of E. coli in the current investigation was 48%. The high prevalence of E. coli isolated from the floor was 61.7%. The prevalence of E. coli isolated from veal meat, tables, walls, and worker’s hand was 60%, 56.7%, 31.7%, and 30%, respectively. A statistically significant difference in contamination rates was observed among sample types (χ²=23.96, p < 0.001). The highest prevalence was recorded in the floor samples (61.7%). No statistically significant difference was observed between the two areas (χ²=0.00, p > 0.05), indicating similar hygienic and contamination conditions in butcher shops on both sides of the city. Additionally, PCR analysis confirmed the presence of the uidA gene in all E. coli isolates (100%), the presence of the Stx1 gene in 73.3%, and the presence of the Stx2 gene in 23.3% of E. coli isolates. Four different gene profiles were found in E. coli. Profile II (uidA + Stx1) accounted for 53.3% of all isolates.

Conclusion: All butcher shops’ equipment spread E. coli, the retail meat was tainted by the bacteria, and the E. coli isolates had the Stx1 and Stx2 genes.

Keywords: Butchers, E. coli, Retail meat, Shiga toxin genes, Shops.

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Introduction

Meat is one of the most valuable sources of nutrition for humans because of its rich composition of essential nutrients. Meat is a crucial source of high biological value protein that provides all of the essential amino acids required for human growth, maintenance, and metabolism. Meat is also a source of critical micronutrients, including iron, zinc, selenium, and B vitamins, particularly vitamin B12, which are important for development, tissue repair, and body function preservation. Numerous zoonotic foodborne microorganisms are capable of producing different kinds of toxins, which can cause food poisoning. The pathogenic bacteria can be transferred from animals to humans through consumers (Neamah et al., 2022). Meat can get contaminated by zoonotic foodborne germs while animals’ carcasses are being dressed, eviscerated, transported, and stored in the abattoir (Awosile et al., 2021). Furthermore, meat is exposed to contaminants, such as pollutants, rodents, and insects, and unsafe conditions during animal slaughter and meat preparation (Laury and Echeverry, 2009; Amniattalab and Ghalandarzadeh, 2019). Foodborne pathogenic bacteria isolated from meat and from numerous human food poisoning cases are of greatest importance (Kalin and Öngör, 2014; Yousif et al., 2021 ; Othman et al., 2023; Sheet et al., 2023).

Escherichia coli has phenotypic characteristics and inhabits both human and animal digestive systems (Tenaillon et al., 2010). In addition, E. coli spreads through food and can cause a number of deadly infections in both humans and animals (Tarr et al., 2005). E. coli has several genes that encode virulence factors, such as uidA and Shiga toxin 1 and 2 genes (Krüger and Lucchesi, 2015). Furthermore, E. coli harboring the uidA gene produces the β-glucuronidase enzyme, which is essential for the hydrolysis of glucuronides into glucuronic acid (Majowicz et al., 2014). Two different types of toxins produced by E. coli are known as Shiga Toxins 1 and 2. Shiga toxin synthesis. This type of E. coli, frequently referred to as Escherichia coli (STEC), is responsible for global outbreaks and isolated cases of STEC illnesses, resulting in a variety of problems (Castro et al., 2019). Escherichia coli can be identified using various techniques, including molecular biology and conventional methods. Conventional methods rely on media (selection and enrichment media), Gram stain, which analyzes the shape and response of E. coli colonies for isolation, and biochemical tests, which use E. coli DNA to identify the bacteria (Lozinak et al., 2016). The molecular biological approaches depend on identifying the target sequence of the specific-species gene and applying them to validate the findings of traditional tests. Molecular biology methods produce simpler, less expensive, quicker, and more precise results, reducing the identification time from several days to just a few hours (Gao et al., 2011).

The present study aims to: (1) isolate and identify E. coli isolated from Mosul City’s butchers’ shops; (2) confirm the E. coli isolates by detecting the uidA gene; and (3) detect the Stx1 and Stx2 genes in E. coli isolates.

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Materials and Methods

Samples collection

In this study, 300 randomly collected samples were taken from veal meat sold at retail and various spots of butcher shops, including workers^, hands, tables, walls, and floors, in various regions on the right (Ras Al-Jada, Zanjili, July 17, Clock area, Al-Mansour, and new Mosul) and left (Sumer, Palestine, North Garage, Al-Sabawi, Algeria, and Prophet Youns) sides of Mosul city, Iraq (Fig. 1). Furthermore, 60 samples were obtained from each worker’s hands, veal meat, tables, walls, and floors on both sides of the city. The study period began in February 2025 and ended in July 2025. Sterile containers were used to collect meat, and swabs were used to collect other samples. The samples were then taken to the Laboratory of Public Health at the College of Veterinary Medicine, University of Mosul, Iraq. All samples were pre-enriched by diving in peptone water and then incubated at 37°C for the entire night.

Fig. 1. Geographical map of Mosul illustrating the sampling points.

Escherichia coli isolation and characterization

Swabs from butcher shops (workers^, hands, tables, walls, and floors) and veal meat samples were used to isolate and identify pathogenic E. coli. After inoculation with the nutrient broth (LAB, UK), all samples and swabs were incubated for 24 hours at 37°C for 24 hours. One loop of nutrient broth was spread onto MacConkey agar (LAB, UK) and EMB for the classical culture method, which was then incubated for 24 h at 37°C. Additionally, the present study used Brilliance E. coli/coliform Agar (Oxoid, UK). The putative E. coli isolates were verified using the IMViC series of biochemical tests (Momtaz et al., 2013). Before being used further for workshop examination, all E. coli isolates were frozen in brain heart infusion broth (15% glycerol) at ?80°C.

DNA isolation

Escherichia coli was cultivated on selective media at 37°C for 24 h. E. coli’s deoxyribonucleic acid was isolated following the directions on the FlaPure Bacteria Genomic DNA Extraction Kit (Genesand Kit, China). A NanoDrop spectrophotometer (Jenway, UK) was used to determine the DNA concentration. The range of DNA concentrations was 30 ng/μl to 60 ng/μl.

Reaction of the PCR

The detection of E. coli genes—including uidA, Stx1, and Stx2—was carried out using polymerase chain reaction (PCR) assays. The following amplicon sizes were expected for every target gene: uidA, 623 bp (Moyo et al., 2007), Stx1, 347 bp (Fujioka et al., 2013), and Stx2, 282 bp (Brian et al., 1992) (Table 1). These gene targets were selected for their relevance in confirming species identity and Shiga toxin production. The PCR reaction was performed in 200 μl PCR tubes (Biozym, Germany) with a total volume of 25 μl. After being amplified, the PCR products were identified by electrophoresis on a 2% agarose gel (Peqlab, Germany). A molecular size marker of 100 bp DNA was used to measure the fragment lengths of the ensuing amplicons. The PCR reaction mixture consisted of 12.5 μl of Master Mix (2×) (Addbio, Korea), 4 μl of E. coli genomic DNA, 1 μl of each primer (1 and 2), and 6.5 μl of DNeasy-free water (Promega Corporation, USA).

Statistical analysis

Statistical analysis was performed using the JMP® 16.1 software (SAS Institute Inc., 2021). The chi-square test revealed a significant variation in the percentage of E. coli isolates across various regions in Mosul City, with a p-value of 0.05.

Ethical approval

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Mosul’s College of Veterinary Medicine under approval number UM. Vet. 2025.072. The animal owners provided their informed consent for the sample to be utilized, and all procedures followed ethical guidelines.

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Results

The colony morphology showed that the E. coli isolates were positive for specific biochemical assays and IMViC tests. According to spectrophotometry, the quantity of genomic DNA extracted varied from 30 µg/l to 60 µg/l (Fig. 2). All E. coli isolates were confirmed to be positive for the species-specific uidA gene, supporting their molecular designation. PCR further supported the results from traditional microbiological techniques.

Table 2 shows that the overall prevalence of E. coli isolated from veal meat sold at retail and butchers’ shops was 48% (144/300). The highest isolation rate was observed from floor samples, which was 61.7% (37/60) of the positive cases. In contrast, the lowest prevalence rates were detected in samples taken from workers’ hands (30%) (18/60). Furthermore, the percentages of E. coli isolated from veal meat, tables, and walls were 60% (36/60), 56.7% (34/60), and 31.7% (19/60), respectively. In addition, the chi-square test was used to evaluate differences in E. coli prevalence among sample types (hands, meat, tables, walls, and floors). The overall prevalence of E. coli among all examined samples was 48% (144/300). A statistically significant difference in contamination rates was observed among sample types (χ²=23.96, p < 0.001). The highest prevalence was recorded in floor samples (61.7%), meat samples (60%), and table surfaces (56.7%), whereas lower prevalence rates were observed on wall surfaces (31.7%) and workers’ hands (30%) (Table 2).

Fig. 2. Comparative DNA concentrations of different E. coli isolates.

Table 1. PCR program and primers for uidA, Stx1, and Stx2 genes detection in E. coli.

Furthermore, this investigation discovered that retail meat and butcher shops on Mosul’s left and right flanks contributed 48% (72/150) of the E. coli samples. Moreover, the highest occurrence of E. coli isolated from veal meat on the right side was 60% (18/30), whereas the lowest incidence of E. coli isolated from workers’ hands was 30% (9/30). The occurrence of E. coli isolated from walls, tables, and floors was 40% (12/30), 53.3% (16/30), and 56.7% (17/30), respectively. Additionally, the largest percentage of E. coli found on the flooring was found on the left side, at 66.7% (20/30). The lower incidence of E. coli found on the walls was 23.3% (7/30). Subsequently, it was discovered that 30% (9/30), 60% (18/30), and 60% (18/30) of the E. coli isolates came from the workers’ hands, veal meats, and tables, respectively. In addition, the results of the present study indicated no statistically significant differences in the isolation rates of E. coli among the various types of samples on the right side. Comparative analysis between the right and left sides of Mosul City showed identical overall prevalence rates (48%), with no statistically significant difference between the two areas (χ²=0.00, p > 0.05), indicating similar hygienic and contamination conditions in butcher shops on both sides of the city (Table 3).

Table 2. E. coli prevalence in retail meat samples and butchers’ shops

Fig. 3. Visualization of the uidA gene (623 bp) amplicon from E. coli isolates using 2% agarose gel electrophoresis. Lane M is DNA markers using a 100-bp ladder from Biozym Diagnostic. Lane 1 contains a positive control, Lane 2 shows a negative control, and Lanes 3–7 display-positive isolates.

Table 3. Comparative prevalence of Escherichia coli isolates from meat and butcher shops in the right and left areas of Mosul City

Table 4 PCR analysis confirmed the presence of the uidA gene in all E. coli isolates (100%) (Fig. 3). In addition, PCR analysis revealed that the Stx1 gene was present in 73.3% (22/30) of E. coli isolates, whereas the Stx2 gene was detected in 23.3% (7/30) (Figs. 4 and 5). Table 5 reveals that the E. coli isolates had four different gene profiles. Profile II (uidA + Stx1) was the most common profile, accounting for 53.3% (16/30) of all isolates. Profile IV (uidA + Stx1+ Stx2), which had the greatest gene-rich profile, was observed in 16.7% (5/30) of the isolates. Profile I (uidA) and Profile III (uidA + Stx2) were observed in 20% (6/30) and 10% (3/30) of the isolates, respectively.

Fig. 4. Stx1 gene (347 bp) amplicon visualization from E. coli isolates using 2% agarose gel electrophoresis. Lane M is DNA markers using a 100-bp ladder from Biozym Diagnostic. Lane 1 contains a positive control, Lane 2 shows a negative control, and Lanes 3–7 display-positive isolates.

Table 4. Frequency and prevalence of uidA, Stx1, and Stx2 in E. coli (n=30).

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Discussion

Many types of foodborne bacteria cause food poisoning through the consumption of contaminated foods. The origin of E. coli from food-producing animals plays a crucial role in human infection, primarily through the ingestion of pathogen-carrying food products that serve as pathogen reservoirs (Busani et al., 2006). The data obtained confirmed the presence and distribution of E. coli in 48% of butchers’ shops. The obtained data were nearly of those reported in a study conducted in Iraq that showed the prevalence of E. coli in butchers and shops was 45% (Hameed et al., 2021).

Moreover, the E. coli occurrence rate on each side of Mosul city was 72%. The results of our study exceeded the findings of other investigations, including the incidence of E. coli isolated from butchers and shops in Mansoura City, Egypt, which was 26.7% (Sallam et al., 2013); in Hawassa City, Ethiopia, where E. coli was isolated from butcher shops and meat samples at a rate of 2.3% (Worku et al., 2022); and in Buenos Aires, Argentina, where the isolation rate was 11.1% (Barril et al., 2019).

In contrast, the current study’s findings were lower than those identified in a previous study conducted in Saudi Arabia, where the isolation rate of E. coli reached 60% (Iyer et al., 2013). The tolerance of bacteria to various physical and chemical surroundings, along with insufficient sanitation and disinfection practices designed to kill them, may be the cause of this variation in E. coli isolation rates. Furthermore, blood, feces, mucus fluids, insufficiently chlorinated water, and contaminated hides offer a perfect environment for E. coli to grow and survive for a long time.

Fig. 5. Stx2 gene (282 bp) amplicon visualization from E. coli isolates using 2% agarose gel electrophoresis. Lane M is DNA markers using a 100-bp ladder from Biozym Diagnostic. Lane 1 contains a positive control, Lane 2 shows a negative control, and Lanes 3–7 display-positive isolates.

Table 5. Genotypic profiles of E. coli isolated from retail meat and butcher shops.

Additionally, 60% of E. coli were isolated from meat. The occurrence of E. coli from meat was greater in the United States (21.1%) (Tate et al., 2021), India (43.1%) (Sethulekshmi et al., 2018), and Egypt (54%) (Gwida et al., 2014) than in the other countries (60%) (Gwida et al., 2014). The incidence of E. coli isolated from meat was lower in South Africa (74.5%) (Vorster et al., 1994) and Burkina Faso (100%) (Kagambega et al., 2011). Differences in meat preparation procedures, national or district factors pertaining to animal feeding schemes, and the methods utilized for microbiological testing could all contribute to the mixture in E. coli infection rates in meat (Rigobelo et al., 2008). A number of variables, including animal skin, meat-cutting equipment, unsanitary conditions, and a drop in worker hygiene, contribute to the contamination of meat and related products with E. coli (Boukary et al., 2012). The majority of the population in impoverished republics choose to purchase low-cost meat from unregulated markets, which do not have hygienic requirements or meat safety protocols (James et al., 2014).

Furthermore, this study found that the prevalence of E. coli in the hands of workers was 30%. Our results were in agreement with those of a study in Saudi Arabia, which recorded the prevalence of E. coli in workers’ hands as 30% (Elabbasy et al., 2021). In addition, our results showed that the prevalence of E. coli from workers’ hands was higher than that in Ghana (10%) (Adzitey et al., 2021) and Ethiopia (16.7%). (Sebsibe and Asfaw, 2020). The prevalence of E. coli in workers’ hands in Morocco was 80%, which was higher than the results of our study (Bahir et al., 2022). There are many reasons why butchers’ hands could be contaminated with E. coli. Being in direct contact with tainted meat or animal internal organs throughout slaughter and cutting is one of the main reasons, since fecal debris and intestinal contents. Poor handwashing practices, such as not using soap, disinfectants, or clean water after handling raw meat or utilizing the restroom, are another factor that leads to bacterial contamination. Additionally, the results of this study found that the prevalence of E. coli in the walls and floor of butchers’ hands was 31.7% and 61.7%, respectively. The floor and walls of butcher shops can become contaminated for a number of reasons. Throughout slaughter or evisceration, intestinal contents and fecal wastes may leak or splash, contaminating surrounding walls and floors, or aerosols created by high-pressure water hose cleaning transmit bacteria from contaminated floors to walls, tools, and meat surfaces. The persistence of E. coli on surfaces can be caused by insufficient cleaning, a lack of effective disinfectants, or a lack of time for sanitizers to come into contact with surfaces.

Furthermore, 73.3% of E. coli had the Stx1 gene and 23.3% had the Stx2 gene. Our results were consistent with further research indicating that the Stx1 gene is more frequently found in E. coli isolates than the Stx2 gene (Ranjbar et al., 2017; Nehoya et al., 2020). Various studies have reported variations in the presence of the Stx1 and Stx2 genes in E. coli isolates; according to one study, 51% of isolates had the Stx2 gene and 29% of isolates had the Stx1 gene (Mora et al., 2007 ). Previous investigations found that 8.8% of isolates had both Stx1 and Stx2, 5.3% of isolates contained Stx1, and 86% of isolates had Stx2 (Llorente et al., 2014), whereas E. coli isolated from Vietnamese meat and fish did not possess the Stx1 or Stx2 genes (Van et al., 2008). The diversity in Stx1 and Stx2 gene occurrence across E. coli isolates can be attributed to host-related, ecological, and molecular characteristics. The observed heterogeneity is a result of differences in phage-integration effectiveness, gene stability, and horizontal gene transmission events. Bacteriophages carry both the Stx1 and Stx2 genes at the molecular level (Franz et al., 2007). Ecologically, gene prevalence depends on the source of isolation (cattle, goats, meat, humans, etc.). For example, some investigations showed that Stx1 was more prevalent in isolates from small ruminants, but Stx2 was more prevalent in strains related to humans or cows (Ndegwa et al., 2020). Lastly, sample type, region, seasonal variations, and animal husbandry techniques are additional factors that influence gene carriage rates. All of these factors work together to explain why certain E. coli populations have stx11ed, others have Stx2, and others have neither.

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Conclusion

This study assessed the sanitation practices of meat and butcher shops on both sides of the city of Mosul. The isolation of E. coli from meat samples that contamination may have occurred at various processing stages, including handling, transportation, storage, or slaughter, most likely under unhygienic conditions. Moreover, the presence of E. coli on the surfaces of butcher shops and workers’ hands underscores their significant contribution to the spread of the bacterium, facilitating contamination of meat and related products. Furthermore, the detection of E. coli on meat and within butcher shops indicates either repeated use of equipment without adequate sanitation or improper washing, cleaning, and sterilization procedures. Such lapses may lead to E. coli contamination of meat, posing a significant health risk to consumers. Unsanitary conditions can facilitate the proliferation of E. coli, enhancing the risk of Shiga toxin production and leading to foodborne diseases among consumers. The E. coli isolates obtained in this study exhibited considerable genetic diversity and carried both Stx1 and Stx2 genes, which was confirmed by the identification of several virulence-associated genes.

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Acknowledgment

Our heartfelt thanks go out to the College of Veterinary Medicine of the University of Mosul for their helpfulness and for providing the facilities and materials required for performing this study.

Funding

The authors declare that no governmental or non-governmental organization provided financial support for this research.

Authors’ contributions

The authors were involved in the collection of samples and laboratory analysis. The authors performed data analysis and interpretation. The third author drafted and revised the manuscript, and approved the final manuscript.

Conflict of interest

The author verifies that there were no conflicts of interest associated with the study’s publication, data analysis, or authorship.

Data availability

The data supporting this study’s findings are available from the corresponding author on reasonable request.

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