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

    Noor A. Alttai Raad A. Al-Sanjary Omar H. Sheet

Iraqi Journal of Veterinary Sciences, 2023, Volume 37, Issue 2, Pages 453-457
10.33899/ijvs.2022.136232.2571

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Abstract

The current study aims to detect the presence of some virulence genes of Escherichia (E.) coli in fish. 46 strains of E. coli were previously isolated from 153 fish samples, including 28 and 18 isolates of E. coli from local markets and fish farms, respectively, in Nineveh Governorate from November 2021 to January 2022. The study results show that all isolates of E. coli possessed the uidA gene with a molecular weight of 623 bp. In addition, they show that 88.9% (16/18) isolates from farmed fish samples possessed the Stx1 gene with a molecular weight of 347 bp, while 72.2% (13/18) of them carried the Stx2 gene with a molecular weight of 589 bp. Also, the study unveils that 89.3 % (25/28) isolated from the market fish samples possessed the Stx1 gene with a molecular weight of 347 bp, and 85.7% (24/28) isolates carried the Stx2 gene with a molecular weight of 589 bp. The rfb gene is detected in this study, neither in farm fish nor in the samples from the local fish markets using the PCR technique. Likewise, it shows that E. coli isolated from fish possessed the Stx1 and Stx2 genes, major causative agents of food poisoning for consumers. Finally, the study gives important information about the application of health conditions on fish farms and fish markets to prevent contamination and reduce infections by foodborne pathogens that cause food poisoning in humans.
Keywords:
    Fish farms Local fish markets E. coli Stx1 and Stx2 genes
Main Subjects:
  • Meat Hygiene

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

 

Gene

Primer

Sequence (5- 3)

Amplicon Size [bp]

Reference

uidA

uidA-1

5-CCAAAAGCCAGACAGAGT-3

623

Moyo et al., 2007

uidA-2

5-GCACAGCACATCAAAGAG-3

Stx1

Stx1-1

5-AGTTAATGTGGTGGCGAAGG-3

347

Fujioka et al.,2013

Stx1-2

5-CACCAGACAATGTAACCGC-3

Stx2

Stx2-1

5- TTCGGTATCCTATTCCCGG-3

592

Fujioka et al.,2013

Stx2-2

5- CGTCATCGTATACACAGGAG-3

Rfb

rfb-1

5-CGGACATCCATGTGATATGG-3

259

Paton et al., 1998

rfb-2

5-TTGCCTATGTACAGCTAATCC-3

 

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

 

Location

Number of the samples

Examine

+ve E. coli

+ve uidA gene

Hawi church

25

4

4

Wana

25

6

6

Hamdaniya

25

8

8

Total

75

18 (24%)

18 (24%)

Al-Medan

28

17

17

Albaladiat

25

6

6

Nabi younis

25

5

5

Total

78

28 (35.9%)

28 (35.9%)

 

 

 

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

 

Location

Number of the samples

Examine

+ve to E. coli

+ve to Stx1 gene

+ve to Stx2 gene

+ve to rfb gene

Hawi church

25

4

3

2

-

Wana

25

6

5

4

-

Hamdaniya

25

8

8

7

--

Total

75

18 (24%)

16 (88.9)

13 (72.2%)

0

Al-Maidan

28

17

15

13

-

Al-Baladiyat

25

6

6

6

-

Nabi younis

25

5

4

5

-

Total

78

28 (35.9)

25 (89.3%)

24 (85.7)

0

 

 

 

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.

  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.
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(2023). Detection of some virulence gene Stx1, Stx2, and rfb of Escherichia coli isolated from fish in Nineveh governorate, Iraq. Iraqi Journal of Veterinary Sciences, 37(2), 453-457. doi: 10.33899/ijvs.2022.136232.2571
Noor A. Alttai; Raad A. Al-Sanjary; Omar H. Sheet. "Detection of some virulence gene Stx1, Stx2, and rfb of Escherichia coli isolated from fish in Nineveh governorate, Iraq". Iraqi Journal of Veterinary Sciences, 37, 2, 2023, 453-457. doi: 10.33899/ijvs.2022.136232.2571
(2023). 'Detection of some virulence gene Stx1, Stx2, and rfb of Escherichia coli isolated from fish in Nineveh governorate, Iraq', Iraqi Journal of Veterinary Sciences, 37(2), pp. 453-457. doi: 10.33899/ijvs.2022.136232.2571
Detection of some virulence gene Stx1, Stx2, and rfb of Escherichia coli isolated from fish in Nineveh governorate, Iraq. Iraqi Journal of Veterinary Sciences, 2023; 37(2): 453-457. doi: 10.33899/ijvs.2022.136232.2571
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  1. Norman RA, Crumlish M, Stetkiewicz S. The importance of fisheries and aquaculture production for nutrition and food security. Rev Sci Tech. 2019;38(2):395-407. DOI: 10.20506/rst.38.2.2994
  2. Sharp JH, Clements K, Diggens M, McDonald JE, Malham SK, Jones DL. E. coli is a poor end-product criterion for assessing the general microbial risk posed from consuming norovirus contaminated shellfish. Front Microbiol. 2021;12:150. DOI: 10.3389/fmicb.2021.608888  
  3. Kamala K, Rajeshkumar S, Sivaperumal P. The predominance of Shiga toxin-producing E. coli in the southeast coast of India. Mar Pollut Bull. 2022;174:113188. DOI: 10.1016/j.marpolbul.2021.113188
  4. Alsanjary LH, Sheet OH. Molecular detection of uidA gene in Escherichia coli isolated from the dairy farms in Nineveh governorate, Iraq. Iraqi J Vet Sci. 2022;36(3):599-603. DOI: 10.33899/ijvs.2021.131046.1913
  5. Othman SM, Sheet OH, Alsanjary LH. Isolation and identification of Escherichia coli O157:H7 isolated from veal meats and butchers’ shops in Mosul city, Iraq. J Appl Vet Sci. 2022;7(4):55-61. DOI: 10.21608/javs. 2022.152213.1164
  6. Petronillah R, Robert K, John V, Nyoni S. Isolation and identification of pathogenic bacteria in edible fish: A case study of Fletcher dam in Gweru, Zimbabwe. Int J Sci Res. 2013;2:269-73. DOI: 10.1.1.680.9981 
  7. Costa RA. Escherichia coli in seafood: A brief Overview. Adv Biosci Biotechnol. 2013;4(3):450-454. DOI:  10.4236/abb.2013.43A060
  8. Gordon D, Cowling A. The distribution and genetic structure of Escherichia coli in Australian vertebrates: Host and geographic effects. Microbiol. 2003;149(12):3575-3586. DOI: 10.1099/mic.0.26486-0
  9. Aleksender N, Margarita T, Inga E, Olga V, Aivars B. Major foodborne pathogens in fish and fish products: A review. Ann Microbiol. 2016;66:1-15. DOI: 10.1007/s13213-015-1102-5
  10. Newaj A, Mutani A, Ramsubhag A, Adesiyun A. Prevalence of bacterial pathogens and their anti-microbial resistance in tilapia and their pond water in Trinidad. Zoonoses Public Health. 2008;55(4):206-213. DOI: 10.1111/j.1863-2378.2007.01098.x
  11. Noor El-Deen AE, Atta NS, Abd El-Aziz MA. Oral vaccination of Nile tilapia (Orechromis niloticus) against motile Aeromonas septicemia. Nat Sci. 2010;8(2):21-25. [available at]
  12. Sanyal S, Banerjee S. Bacterial population of fish and their environment in ponds utilized for natural purification of wastewater. Appl Ecol Environ Sci. 2021;9(7):649-655. DOI: 10.12691/aees-9-7-4
  13. Nagamatsu K, Hannan TJ, Guest RL. Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection. Proc Natl Acad Sci USA. 2015;112(8):871-880. DOI: 10.1073/pnas.1500374112
  14. Spinale JM, Ruebner RL, Lawrence C. Long-term outcomes of Shiga toxin hemolytic uremic syndrome. Pediatr Nephrol. 2013;28:2097-2105. [available at]
  15. Galal HM, Hakim AS, Sohad M. Dorgham. Phenotypic and virulence genes screening of Escherichia coli strains isolated from different sources in delta Egypt. J Life Sci. 2013;10(2):352-361. [available at]
  16. Wogu MD, Maduakol M. Evaluation of microbial spoilage of some aqua cultured fresh fish in Benin city, Nigeria. Ethiop J Environ Stud Manag. 2010;3(3):18-22. DOI: 10.4314/ejesm.v3i3.63960
  17. Fouz B, Toranzo AE, Milan M, Amaro C. Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae. J Appl Microbiol. 2000;88:531-535. DOI: 10.1046/j.1365-2672.2000.00992.x
  18. Lokollo E, Mailoa MN. Handling techniques and microbial contamination of fresh flying fish in traditional markets in Ambon city. J Pengolah Has Perikan Indones. 2022;23(1):103-11. [available at]
  19. Brown AE, Smith HR. Benson's microbiological applications, laboratory manual in general microbiology. 14th ed. New York: McGraw Hill Higher Education; 2017: 438 p.
  20. Moyo SJ, Matee MI, Largeland N, Mylvaganam H. Identification of diarrheagenic Escherichia coli isolated from infant and children in Dares Sallaam, Tanzania. BMC Infect Dis. 2007;7:92. DOI: 10.1186/1471-2334-7-92
  21. Dashti AA, Mehrez MJ, Abdulsamad MA, Hussein MD. Heat treatment of bacteria: A simple method of DNA extraction for molecular techniques. J Kuwait Med Assoc. 2009;41(2):117-122. [available at]
  22. Cardozo MV. Pathogenic Escherichia coli in wild fish and intensive fish farming for human consumption. J Microb Biochem Technol. 2013;5(4):79. DOI: 10.4172/1948-5948.S1.010
  23. Ashraf AA, El-Hofy F, El-Gamal AM, Ibrahim HO. Bacteriological and molecular studies on bacteria transmitted from fishes to human. Int J Adv Res. 2018;6(1):1047-1059. DOI: 10.21474/IJAR01/6319
  24. Taha ZM, Yassin NA. Prevalence of diarrheagenic Escherichia coli in animal products in Duhok province, Iraq. Iran J Vet Res Shiraz Univ. 2019;20(4):255-262. [available at]
  25. Siddhnath K, Majumdar RK, Parhi J, Sharma S, Mehta NK, Laishram M. Detection and characterization of Shiga toxin-producing Escherichia coli from carps from integrated aquaculture system. Aquac. 2018;487:97-101. DOI: 10.1016/j.aquaculture.2018.01.008
  26. Ribeiro LF, Barbosa MC, de Rezende Pinto F., Guariz CL, Maluta RP, Rossi JR, Rossi GM, Lemos MF, do Amaral LA. Shiga toxigenic and enteropathogenic Escherichia coli in water and fish from pay-to-fish ponds. Appl Microbiol. 2015;62:216-220. DOI: 10.1111/lam.12536
  27. Kumar HS, Otta SK, Karunasagar I. Detection of Shiga-toxigenic Escherichia coli (STEC) in fresh seafood and meat marketed in Mangalore, India by PCR. Lett Appl Microbiol. 2001;33(5):334-338. DOI: 10.1046/j.1472-765X.2001.01007.x
  28. Ritter L, Solomon K, Sibley P, Hall K, Keen P, Mattu G, Linton B. Sources, pathways, and relative risks of contaminants in surface water and groundwater: A perspective prepared for the Walkerton inquiry. J Toxicol Environ Health. 2002;65:1-142. DOI: 10.1080/152873902753338572
  29. Simpson JM, Santo-Domingo JW, Reasoner DJ. Microbial source tracking: State of the science. Environ Sci Technol. 2002;36:5279-5288. [available at]

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