Detection of some virulence gene Stx1, Stx2, and rfb of Escherichia coli isolated from fish in Nineveh governorate, Iraq

1. Isolation and Identification of E. coli from farm and local market fish.
2. Confirmation of these isolate using the uidA genes.
3. Detection of the virulence gene stx1, stx2, and rfb from these E. coli isolate.

Introduction

Fish is one of the important foods for humans, because it is rich in easily digestible proteins and has a high nutritional value. Yet, it is characterized by being a perishable food product, which leads to a decrease in its quality, especially during treatment and storage, a thing that limits its life span (1). Several researchers recently indicated that these bacteria were isolated from mollusks (oysters) found in polluted coastal waters (2). In addition, E. coli were isolated from fish found in coasts contaminated with sewage water (3). Also, E. coli was isolated from different animals such as cows and their environment (4) and from meat and the butchers’ shop (5). Freshwater fish may contaminate with various types of pathogenic microorganisms from the various sources, which are usually either primary sources through the presence of these fishes in the aquatic environment or secondary sources during the harvesting of these fishes, as well as from transportation, marketing and storage, which lead to the pollution of these fishes and make them unsuitable for human consumption or to be carriers of some pathogens to humans (6). Therefore, the low count of bacteria and decreased contamination with pathogens bacteria in fresh fish indicate that fish is suitable for human consumption and fish is of high quality (7). The offal of fish such as intestines, scales, fins, gills and the bad handling of them play an important role in the spread of harmful bacteria, which leads to cause hygienic problems. Although the presence of some types of microorganisms such as E. coli bacteria in fish is uncommon (8), these pathogenic microorganisms were linked to food-borne diseases through food routes from eating fish or fish products (9). Enterobacteriaceae are widespread in all parts of fish due to the imbalance between the bacteria and the host environment (10). This family of bacteria is a major contaminant and pathogen in farmed fish (11), but in most cases, these microorganisms are considered part of the normal microorganisms of fish. They are opportunistic bacteria that can cause some diseases when colonizing human body (12) such as urinary tract infections as well as causing hemorrhagic colitis, acute renal failure, irritable bowel syndrome or death (13,14). There are different strains of E. coli such as EPEC, ETEC, EHEC, STEC, which give an indication of wastewater contamination, but the Shiga toxin-producing E. coli (STEC), in addition to the enteric pathogen (EPEC) are considered important pathogens due to their zoonotic origin that transport diseases to humans, especially in developing countries through consuming the contaminated fish (15, 16).

 Selling fresh fish in traditional fish markets or retailers, or even farm-raised fish, which do not apply hygienic conditions during the breeding of fish may contaminate fish with pathogenic bacteria that were originally in water (17). Therefore, fish traders in the markets paid attention to how to deal with these foods in terms of transportation, storage, and used clean water to wash fish fielded in traditional fish markets (18). Henceforth, this study aims at detecting the presence of some virulence genes of E. coli in fish in fish farms and local markets in Nineveh Governorate.

Materials and methods

A total of 46 E. coli isolates were previously isolated from 153 fish samples, which included 28 isolates of E. coli from local markets and 18 isolates from local markets and fish farms from various regions in Nineveh Governorate from November 2021 to January 2022. The samples were directly transferred to the laboratories of the Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Iraq, for processing, isolation and the molecular detection of E. coli bacteria (19, 20).

Ethical approve OR data collection permit

University of Mosul, College of Veterinary Medicine, the approval issue number and date are 1650 at 21/11/2021.

DNA Extraction and Amplification

The selected colonies were placed to 200 μl of sterile distilled water in a 1.5 mL Eppendorf tube (21). Then, they were mixed with a vortex mixing device. Cells were lysed for at least 15 seconds and DNA extracted using a laboratory kit prepared by (Jena) Bioscience (22). The conventional species-specific polymerase chain reaction (PCR) technology was used to confirm E. coli isolates using uidA universal primers and the virulence factors (stx1, stx2, and rfb) (Table 1).

Table 1: Primers Sequence of Virulence Gene (uidA, Stx1, Stx2, and rfb) of E. coli

The master mix for PCR reaction was prepared using the Gen Net Bio kit by calculating the required volumes of reaction components for each sample. The additives were mixed well and dispensed in a volume of 20 μl (12.5 μl master mix, 1 μl for each primer and 5.5 μl of Nuclease-Free Water) put into 0.2 ml PCR tubes for amplification. Then, 5 μl of DNA extracted from each sample were added separately to each tube to reach the total volume of 25 μl. The PCR reaction was carried out in a T100TM thermocycler (Bio-Rad, USA), using the following parameters for all genes used in this study at 95°C for 5 min as initial denaturation, then for all, the final elongation was 72°C for 5 min, but the rest of program was, for uidA: (94°C - 1 min, 57°C - 1 min, and 72°C - 1 min) and for stx1: (94°C - 30s, 57°C - 30s, 72°C - 30s), for stx2: (94°C - 30s, 55°C - 30s, 72°C - 30s), and for rfb: (94°C - 30s, 52°C - 30s, 72°C - 30s) for 35 cycles for each. The tubes were removed from the apparatus and placed in a refrigerator at 4-8°C until Gel electrophoresis was performed to confirm the size of amplified products for the genes used in this study.

Results

The results of the current study declared that all isolates of E. coli isolated from fish samples, from both farmed fish and local market fish were positive for the uidA gene with a molecular weight of bp 623 (Table 2 and Figure 1).

Table 2: Number of Positive Samples of E. coli Isolate and uidA Gene in all Study Areas

Figure 1: Shows electrophoresis of PCR products for E. coli isolates, where lane C+ represents the positive control, lane M represents Ladder DNA 100bp, lanes 3, 4, 5, 6, 7 represent the positive samples of E. coli uidA gene with a molecular weight of 623 bp and lane C- represents the negative control.

Detection the Stx1, Stx2 and rfb gene

The results of the current study showed that the number of E. coli isolated was 18 isolates from farmed fish samples. In addition, this study found that E. coli isolated from fish farm having the stx1 gene was 88.9% (16/18) with a molecular weight of 347 bp, while E. coli isolated from fish market samples having the stx1 gene was 89.3% (25/28).

The results of the current study found that E. coli isolated from farmed fish samples having the Stx2 gene was 72.2% (13/18) with a molecular weight of 589 bp. Moreover, E. coli isolated from the local fish market samples having the Stx2 gene was 85.7% (24/28) with a molecular weight of 589 bp, while we didn't detect the rfb gene in E. coli isolated from fish farm and local fish markets by using polymerase chain reaction and its primers (Table 3).

Table 3: Detection the Stx1, Stx2 and rfb gene in E. coli isolates

Figure 2: A and B: the virulence factor Stx1 with a molecular weight of 347 bp and Stx2 virulence factor with a molecular weight of 592 bp, where lane 1 represents the positive control, lane 2 represents the Ladder DNA 100 bp, lane 3 represents the negative control, and lanes 1, 2, 3, 4 and 5 represent positive samples.

Discussion

In recent decades, the consumption of fish and its products increased because fish has vitamins, minerals, and fat that are important for humans. Modern techniques are used for detecting the virulence factors encoding genes, which are responsible for producing several types of toxins that cause food poisoning in humans. The previous studies revealed that E. coli can produce the Stx1 and Stx2 genes that cause problems such as high morbidity, high mortality rate of humans, high cost of therapeutics. In addition, they unveiled that E. coli has the ability to resist different types of antibiotics via E. coli possession of the specific gene, which prevents the activity of antibiotics on bacteria (22,23). The results showed that the number of samples that gave positive results for the Stx1 gene from farmed fish was 18 samples with a rate of 88.9%, while the number of samples Stx2 was 15 samples with a percentage of 72.2%. As for the number of samples that gave positive results for the Stx1 gene from the local fish market, it was 25 samples at a rate of 89.3%. As for the Stx2 gene, the number of samples was 24 samples at a rate of 85.7%, while these results of Stx1 and of Stx2 were not detected and do not identify with another study conducted in Duhok Province/ Iraq (24), as well, the results of this study were higher than the results of Siddhnath and colleagues (25), when they revealed the percentage 27% of the positive Stx genes detected by direct PCR. In his research, which he conducted on 18 fish samples, where 8 samples contained Sxt1 by 44% and 14 samples contained Stx2 by 77%, Ribeiro et al. (26) revealed that out of the 115 isolates, 2 (1.74%) were positive only for the Stx1 gene and six isolates 5.21% for Stx2 gene. The isolates positives for Stx2 were found in water and fish gastrointestinal tract samples, while there are no virulence genes detected in skin. In our study, in which the encoding of the O antigen was used for the detection of O157 serotype, we did not detect the rfb genes. This result is in harmony with that yielded in another study, where no detection is made (27).

Effluents from slaughterhouses and municipal wastewater treatment, as well as sewage, leakage of sewage systems, uncontrolled discharge of feces and excreta from wildlife and runoff of manure and fecal residues deposited in fields and pastures, may pollute the water and thus contaminate the fish that live in this water (28,29).

Conclusion

Fish contaminated with E. coli is deemed a major cause in food poisoning for humans. The fish market applying poor methods to clean and store fish help to grow and multiply bacteria, which leads to cause food poisoning. Importantly, E. coli has the Stx1 and Stx2 genes that can synthesize the stx1 and stx2 toxins, which may be transmitted to humans while consuming contaminated food with E. coli that causes health problems. E. coli isolates used in this study did not contain the rfb gene encoding E. coli O157:H7. Finally, the PCR technique is regarded as a modern method to detect E. coli based on the target sequence of the gene.

Acknowledgments             

The authors express their gratitude for the efforts of College of Veterinary Medicine, University of Mosul, in granting them all possible facilitations.

Conflict of interest

The researchers confirmed that there is no conflict of interest.