Iraqi Journal of Veterinary Sciences

Introduction

Many Zoonotic foodborne microorganisms are transmitted from animals and their products to consumers while consuming the contaminated foods (1). The Zoonotic foodborne microorganisms have been able to contaminate meat while processing of carcasses in the abattoir (2). The most critical foodborne microorganisms isolated from meat and its products were Escherichia coli (E. coli), Salmonella spp., Listeria monocytogenes, and Campylobacter spp., which isolated from several cases of outbreaks of human illness (3-5). The most important source of proteins for human beings is meat which may be exposed to contamination by E. coli during the slaughter of the animals under unhygienic conditions and slaughter practices, pollutants, insects, and rodents (6,7). E. coli is a Gram-negative, rod-shaped, facultatively anaerobic, which lives in the intestines of animals and humans (8). Humans may be infected with pathogenic Shiga toxin-producing E. coli (STEC) which causes various types of disease and sometimes death (9,10). STEC can cause outbreaks and food poisoning worldwide due to consuming contaminated food like meat (meat products) and milk (dairy products) (11,12), water, fruits, and vegetables (13). In addition, equipment such as knives, tables, saws, hooks, and other utensils may play a significant role in spreading the STEC during processing carcass and meats cuts (14). STEC can produce the Shiga toxins (15). The Shiga toxins (Stx1 and Stx2) are the primary virulence genes (16). Stx1 and Stx2 are responsible for causing diseases in consumers and animals (17). The methods used to identify the STEC are based on the classical methods including the selective media, chromogenic media, and biochemical tests, also on the Molecular biology techniques such as polymerase chain reaction (PCR) and RT-PCR used to detect the target sequence gene (18). In the last decades, few studies have been performed to identify the STEC and its toxins isolated from meat. Therefore, the project was carried out to determine the prevalence of STEC in meats and butcher shops in Mosul city, Iraq.

 The study aimed to identify the phenotypic characterization of E. coli isolated from meats and butcher shops by using selective media, chromogenic media, and biochemical tests, as well as to identify the genotypic characterization of E. coli by detecting the uidA, Stx1, and Stx2 gene by using the PCR assay.

Materials and methods

Ethical approve

Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, issue number 1418, 21.10.2021.

Sampling

In the current study, 504 samples were collected randomly from meats and different parts of a butcher’s shops (84 samples from each knife, hooks, tables, machines, worker's hands, and veal meat) of the right and the left part of Mosul city. The study period started in September 2021 and ended in January 2022. Meats were collected using sterile containers, while other collected samples were gathered using swabs which were put in containers (sterile) and then transported to the Researchers Center for diagnosis of zoonotic pathogenic bacteria.

E. coli isolation and identification

Meat samples and butcher shops parts swabs were analyzed to isolate and identify of pathogenic E. coli. All samples and swabs were inoculated in the nutrient broth (LAB, United Kingdom) and then incubated overnight at 37°C. For the classical culture method, one loop of nutrient broth was plated on the Eosin Methylene Blue Agar (EMB), and MacConkey agar (LAB, United Kingdom) and then incubated overnight at 37°C. In addition, Brilliance E. coli/coliform Agar (Oxoid, United Kingdom) was used in this study to differentiate between generic E. coli and coliform. The suspected E. coli isolates were confirmed using biochemical tests such as Gram stain, Indole test, Methyl Red test, Citrate Utilization test, and Voges-Proskauer test, Catalase, Oxidase, and Triple Sugar Iron agar (19). All the E. coli isolates were frosted in Nutrition broth (15% glycerol) at -80ºC until further use.

DNA isolation and template production

The suspected E. coli were cultured on the Brilliance E. coli/coliform agar overnight at 37°C. Deoxyribonucleic acid of E. coli was isolated according to the instructions of the DNeasy Blood and tissue kit (Geneaid, Korea). The concentration of Deoxyribonucleic acid of E. coli was estimated using the Bio-drop device then and stored at -20°C for further analysis.

UidA, Stx1, and Stx2 genes amplification

The PCR assay, amplified the sequence of the uidA, Stx1, and Stx2 of E. coli bacteria isolated (Table 1). Twenty-five μL (total volume of PCR reaction) (12.5 μL of 2×Go Taq Green Mix Master including (Promega Corporation, USA), 1 μL of primer-F, 1 μL primer-R, 6.5 μL of nuclease-free water (Promega Corporation, USA), and (v) 4 μL DNA template of E. coli. The whole mixture was put in the Eppendorf tube at 200 μL (Biozym, Oldenhorf, Germany). Finally, the amplicons of the target sequence were determined by using gel electrophoresis, DNA marker 100 bp (ladder), and 2% agarose gel (Peqlab, Erlangen, Germany).

Table 1: PCR program and Primers for detection of uidA, Stx1, and Stx2 in E. coli

PCR program: I=35 times (94°C - 30s, 57°C - 30s, 72°C - 30s), II=35 times (94°C - 30s, 55°C - 30s, 72°C - 30s)

Results

According to the morphology of colonies, the positive E. coli isolates appeared color with a metallic green sheen on the EMB agar, dark pink color on the MacConkey agar, and purple color on the Brilliance E. coli / coliform Agar. In addition, all isolates of E. coli were positive for the special biochemical tests used for identifying the isolates of E. coli. Our findings showed that the prevalence of E. coli in meat and butcher shops was 27.4% (138/504). Furthermore, the percentage of E. coli was higher in meat, 41.7% (35/84). In contrast, the percentage of E. coli was lower in hooks in butcher shops, 16.7% (14/84). Additionally, the percentage of E. coli isolated from knives, tables, worker hands, and machines was 31% (26/84), 31% (26/84), 23.8% (20/84), and 20.2% (17/84), respectively (Table 2).

Table 2: Number and percentage of positive E. coli isolated from meats and butchers’ shops

In addition, our result showed that the percentage of E. coli isolated from meats and butchers’ shops on the right side of Mosul city was 31.9% (65/204) which is more than on the left side 24.3% (73/300). Furthermore, the prevalence rate of the E. coli isolated from knives, tables, machines, workers hands on the right side was 41.2%, 35.3%, 29.4%, and 29.4% that were higher contaminated from the knives, tables, machines, workers' hands on the left side of the Mosul city (Table 3).

Table 3: The percentage of positive E. coli isolated from meats and butchers’ shops on the right and left banks of Mosul city

 According to table 4, our findings showed that all E. coli isolates are possessed the specific species uidA gene. The results of the culture method were confirmed using the PCR technique. The results of the culture method and the results of the PCR assay were concorded (Figure 1). In addition, the Stx1 gene was detected in E. coli isolates 30/138 (21.7%) (Figure 2), while the Stx2 was detected in E. coli isolates 17/138 (12.3%) (Figure 3). Our study also revealed that most E. coli isolates possessed the Stx1 and Stx2 genes 91/138 (66%).

Table 4: The variation rate of the uidA, Stx1, and Stx2 genes in E. coli isolates

Figure 1: UidA gene product in E. coli isolates (623 bp).

Figure 2: Stx1 gene product in E. coli isolates (347 bp).

Figure 3: Stx2 gene product in E. coli isolates (592 bp).

Discussion

In recent decades, E. coli has been considered the prime etiology of food poisoning worldwide. The food animal originated significantly transmitted E. coli to humans via the consuming contaminated foods with E. coli (22). our results demonstrate that the percentage of E. coli isolated from meat and butcher shops was 27.4% (138/504). In addition, this study showed the percentage of E. coli was rising in meat at 41.7% (35/84), which agreed with the previous surveys that found the percentage of E. coli in meat was 43.1% in India (23), 43.4 % in the United States (24). However, our results are lower than the previous surveys that appeared the percentage of E. coli from meat in Egypt was 54% (27/50) (25), 74.5% in South Africa (26), and 100% in Burkina Faso (27). In addition, our results were higher than other studies which found the prevalence rate of E. coli in meat was 1.5% in Iran (28), 17.8% in Australia (29), and 21.1% (49/232) in the United States (30). Various contamination rates of E. coli in meats may be due to variations in the national or geographic features of animal feeding systems, differences in meat processing, and differences in the methods used in the microbiological tests (31). Many studies found that raw and processed meat was more contaminated with E. coli (32). Meat and its products are exposed to contamination with E. coli from entering the animals to the slaughterhouse until consuming meat. Many factors contribute to transmitting E. coli to meat and its products such as the skin of animals, cutting tools and machines, an unhygienic environment, loss of workers' hygiene (33). In developing countries, most people want to buy cheap meats from the informal markets which do not apply hygiene instructions and safety standards when cutting meat (34).

Additionally, the current study showed that E. coli possesses the Stx1 gene in 30/138 (21.7%), while E. coli has the Stx2 gene in 17/138 (12.3%). Also, our study revealed that most E. coli isolates possessed the Stx1 and Stx2 genes in 91/138 (66%). The Stx1 gene is more predominantly detected than Stx2 in E. coli isolates. Our results were similar to previous contributions, which showed that the Stx1 was more frequently found in E. coli (35,36). Many studies showed a difference between the frequent found of the Stx1 and Stx2 genes in E. coli isolates. The previous study showed that 29% of isolates possess the Stx1 gene, 51% isolates carry the Stx2 gene, and 20% possess both Stx1 and Stx2 (37). The previous studies showed that 5.3% of isolates carried Stx1, 86% of isolates had Stx2, and 8.8% of isolates possessed Stx1 and Stx2 (38), another study declared that 40.68% of E. coli isolates contained Stx1 and 5.88% of E. coli isolated have Stx2 (36). At the same time the Stx1 and Stx2 genes did not detect E. coli isolated from meat and fish in Vietnam (39).

Conclusion

Escherichia coli is significant foodborne microorganismin humans due to consuming meats and meat products. Our findings revealed that E. coli was isolated from different samples (Knives, Hooks, Tables, Machines, and Worker Hands) which have a role in spreading the E. coli isolates in meats and meat products. The meat was the most contaminated with E. coli due to passing meat in different contamination stages starting from slaughterhouse until sale meats in the supermarket and meat shops. In addition, the molecular characteristics of E. coli isolates have appeared that some isolates possess only the Stx1 gene, while anther E. coli possess only the Stx2 gene, and most E. coli possess the Stx1 and Stx2 genes together.

Acknowledgments             

The authors express their gratitude for the efforts of the College of Veterinary Medicine, and the University of Mosul to provide them with all facilities.

Conflict of interest

There are no conflicts of interest.

1. Isolation and identification of E. coli isolated from meat and butchers’ shops.
2. Detection of the phenotypic and genotypic characterization of E. coli.
3. Detection of the uidA, Stx1, and Stx2 genes in E. coli.
4. Classical and molecular methods have been used to identify E. coli.

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