Isolation and molecular identification of Escherichia coli strain from fish available in farms and local markets in Nineveh governorate, Iraq

1. Isolation and Identification of E. coli from the farm and local market fish.
2. Confirmation of these isolates by using the uidA genes.
3. Detection of a positive result of serotype (O157:H7) from this isolate.
4. Determination of the resistance of these isolates to antibiotics.

Introduction

The safety of aquatic life, especially fish, is one of the most critical public health issues directly related to agriculture and healthy food production, as is the case with other types of food (1), therefore fish plays a significant role in providing nutrients foods to many animals as well as humans. Sixty percent of fish contribute to the global protein supply, and the developing countries derive more than 30% of animal protein from fish (2), whereas the provision of healthy fish products is necessary from a food safety point of view in order to maintain the health of the consumer because the health of the consumer is part of the safety of the food consumed (3). Besides being a food source, fish also protects humans from diseases prevalent worldwide through daily fish consumption, preventing human heart disease (4). Aquatic organisms, especially fish, contain the many pathogens they transmit to humans. These diseases associated with consuming fish and sea foods, significantly contaminated with microbial pathogens, have increased when they are produced under poor sanitary conditions. Therefore, the contamination of fish causes many risks to consumers' health (5). The pollution of the aquatic environment of fish with sewage water from homes and toxic waste from factories leads to fish contamination with industrial metals resulting from factory waste thrown into running water (6-8). Moreover, bacterial contamination resulting from surface contact with food, a significant source of many foodborne diseases, affects public health through the transmission of microbes from the equipment surfaces to processed foods while dealing with fish during fishing, cleaning, and removing their guts that contaminate fish meat. Additionally, during storage, especially the use of crushed ice in preserving fish until they reach the market, which contains large numbers of disease-causing bacteria that poses a potential threat to public health through the consumption of contaminated fish products and seafood which leads to the food poisoning to consumers (9,10). One of the most important bacterial contaminants is E. coli, which is generally used as an indicator of fish contamination and spoilage. Researchers have conducted several studies about E. coli bacteria in fresh fish and their ready-to-eat products in sales markets (11). E. coli are characterized by their production of toxins, especially Shiga toxin, which leads to food poisoning in the consumer due to eating fish contaminated with bacteria or their toxins (12).

The study aimed to detect the presence and spread of E. coli in fish available on farms and local markets and determine the resistance to antibiotics of these isolated species of E. coli.

Material and methods

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.

Sampling

One hundred and fifty-three fish samples (Cyprinus carpio) were collected in the current study, including: 75 fish samples collected from various fish farms (Hawi Church area, Wana sub-district, and Hamdaniya district) and 78 fish samples collected from different local markets in the Mosul city (Al-Midan area, Albaladiat, and Nabi Yunus markets) at the period from November 2021 to January 30, 2022.

Isolation of bacteria

The isolation of these bacteria was done in the laboratory of the Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Iraq. Based on the conventional methods, the swabs were taken from the fish samples' skin using sterile swabs, placed in tubes containing peptone water, and incubated for18 - 24 h at 37°C as pre-enrichment. Then 1 ml of peptone water was taken and transferred to test tubes containing 9 ml of MacConkey broth and incubated at 37°C for 24 h. From these selective enrichment medium tubes, 1-2 Bacteriological loops were taken and cultured on Eosin-methylene blue agar (EMB), MacConkey agar, Brilliance E. coli/coliform agar, and lastly cultured on Chrome agar E. coli O157:H7 to show the brilliant metallic sheen, pink colonies as lactose -the fermenting characteristic for E. coli, purple colonies and to isolate E. coli O157:H7, and all cultured Petri dishes are incubated at 37°C for 18-24 h (13). To identify these isolates of E. coli, standard biochemical reaction tests (IMVC) are used, including Indole production, citrate utilization test, methyl red, Voges-Proskauer test, oxidase and catalase test, urease and typical reactions on the triglyceride sugar iron agar (14).

Confirmation of E. coli isolates by conventional PCR

The phenotypic-identified E. coli was confirmed by PCR assay to amplify the uidA gene by using PCR that encodes the B-glucuronidase enzyme, which is common in all E. coli species. According to Moyo (15), a 25 μl PCR mixture consisted of 12.5 μl of hot start premix,1 μl of each (10 pmol) of primer F: 5-CCAAAAGCCAGACAGAGT-3 and R:5-GCACAGCACTTCAAAGAG-3, 4 μl of sample DNA, and the remained was filled with nuclease-free water. PCR amplification was carried out in PCR system 9700 GeneAmp with pre-PCR heating at 95°C for 5 min, subsequently exposed to 35 cycles (1 min at 94°C, 1 min at 58°C, 1 min at 72°C), and a final cycle for 5 min at 72°C. The amplified product was run at 85 Volt for 40 min. The amplified PCR products 623 bp in 2% agarose gel prepared in 1x TAE buffer and stained by red safe DNA staining solution were verified.

Antibiotic sensitivity test

The sensitivity of bacteria to antibiotics was tested based on the method (16), and this is done by transferring about 5-6 pure bacterial colonies from the selective media by the bacteriological loop and cultivated in tubes containing 5 ml of peptone water and then incubated for 5 h at a temperature of 37°C (17). Sensitivity test was conducted using 12 types (tetracycline, gentamicin, cephalothin, erythromycin, ciprofloxacin, trimethoprim, ceftriaxone, amoxicillin, streptomycin, nitrofurantoin, cefixime, chloramphenicol) of antibiotics belonging to different groups of antibiotics which obtained as ready-made discs from Bioanalyse Company. A sensitivity test of these isolates on Muller Hinton agar was carried out using the disc diffusion method. (18).

Results

The results of the current study showed that the number of isolates of E. coli bacteria isolated from fish samples, which numbered 75 fish samples from fish farms was 18 positive samples, at a rate of 24%, and 78 fish samples from local markets, 28 positive samples with a percentage of 35.9% as shown in table 1. This study showed that all E. coli bacteria isolated from fish samples, whether farmed or local market fish, possessed the uidA gene with a molecular weight of 623 bp (Figure 1).

Table 1: Number and percentages of isolates of E. coli from fish samples of local market and fish farm

Figure 1: 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.

The number of samples that showed a positive result for the serotype E. coli bacteria (O157:H7) was 7 (3.9%) of the farmed fish samples, while its percentage in the local market fish samples was 11 (14.1%) as present in table 2.

Table 2: Number of positive E. coli O157:H7 isolated from farmed fish and local marked fish

The different results of the resistance of E. coli to the antibiotics used in our study showed in (Table 3) and (Figure 2). The highest sensitivity of E. coli bacteria to the antibiotic ciprofloxin, trimethoprim, and gentamicin was 96, 94, and 86%, respectively. In comparison, the highest percentage of resistance of E. coli to the antibiotics cephalothin, tetracycline, erythromycin, and amoxicillin was 100, 64, 64, and 62%, respectively.

Table 3: Number of resistance and sensitivity of isolates E. coli to the antibiotics used in this study

Figure 2: Resistance and sensitivity of isolated E. coli to the antibiotics collected from local markets and farmed fish.

Discussion

In the past decades, fish farming has been directed to fill the shortage in the provision of animal protein (19). Safe handling of food is an important issue due to the transmission of microorganisms from fish to humans (20). One of these microorganisms is the family of Enterobacteriaceae, usually found in fish's skin and digestive system due to the pollution that occurs in the aquatic environment in which these fish live (21,22).

The current study showed that the percentage of E. coli bacteria isolated from 75 fish farms and 78 samples of local fish markets was 24 and 35.9%, respectively. Aynadis and Engdawork (23) in Southern Ethiopia revealed that the isolation of E. coli done by taking swab samples from the skin of Cyprinus carpio was 32.5%. Our study's results also agreed with Taha and Yassin's findings (24) when they studied Cyprinus carpio fish in Dohuk province, Iraq, where they obtained 39.1%.

The current study showed a positive result for E. coli with serotype (O157:H7) with 16% and 25.6% for both farmed and market fish samples, respectively. Although various methods were developed to detect this particular serotype, standard bacteriological methods remain the gold standard (25). Also, all E. coli positive isolates showed that they had their specific uidA gene, which was detected by using the PCR technique because these isolates of E. coli contained in their DNA. The molecular method was used to detect the species-specific uidA gene in E. coli isolates and to confirm the results of the classical methods (26,27). In order to prevent fecal contamination in fish farms and the names sold in local markets, the results of this study provide useful introductory information that must be used to reduce health risks to consumers.

Antibiotics have been widely used in aquaculture to prevent infection and economic loss. However, the indiscriminate use of antibiotics has led to the emergence of resistant strains, a hazardous situation for consumers due to the transmission of bacterial resistance to humans (28-30). The highest sensitivity of E. coli bacteria to the antibiotic ciprofloxin, trimethoprim, and gentamicin was 96%, 94%, and 86%, respectively. In contrast, the highest percentage of resistance of E. coli to the antibiotic Cephalofen, Tetracycline, Erythromycin, and Amoxicillin was 100, 64, 64, and 62%, respectively. In India, the study of Chakravarty and coworkers (31), his results agreed somewhat with these results, where the antibiotic resistance of E. coli was 100% to Penicillin-G, Tetracycline, and Ampicillin, while 100% sensitive to chloramphenicol, nalidixic acid and ciprofloxacin. On the other hand, Ryu et al. (32) confirmed that commercial fish in retail markets in Seoul, Korea, may constitute reservoirs of multi-antibiotic-resistant bacteria, high resistance to tetracycline 30.7%, cephalotin 11.7%, ampicillin 6.7% and low resistance to ticarcillin 6.1% in strains of E. coli isolated from fish.

Conclusion

In conclusion, we isolated severalE. coli associated with intestinal bacterial infection from fish collected from fish farms and local markets in Mosul city. The presence of the O157:H7 serotype of E. coli in the isolates indicated that these fish were contaminated, and these contaminants may be transmitted to consumers. The results of this study provide useful introductory information that must be used in the proper management of these environments in order to prevent fecal contamination in fish farms and the names sold in local markets, thus reducing health risks to consumers.

Acknowledgments             

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

Conflict of interest

The authors declare that there is no conflict of interest.