Research Article

Detection and Distribution of Escherichia coli O157:H7 Isolated from local Fresh Beef Sold in Baghdad City

Ibrheem E. Ibrhim*, Zina Saab Khudhir

Department of Veterinary Public Health, College of Veterinary Medicine, University of Baghdad, Iraq.

Received | March 04, 2025; Accepted | May 25, 2025; Published | June 27, 2025

*Correspondence | Ibraheem E. Ibrhim, Department of Veterinary Public Health, College of Veterinary Medicine, University of Baghdad, Iraq; Email: [email protected]

Citation | Ibrhim IE, Khudhir ZS (2025). Detection and distribution of Escherichia coli O157:H7 isolated from local fresh beef sold in Baghdad city. J. Anim. Health Prod. 13(3): 539-545.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.3.539.545

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Foods from animal origin, such as chicken, beef, milk, and dairy products, have high protein content, which is essential for the growth and development of the body. However, they transmit various pathogenic and food-spoiling microorganisms which are human health hazards, because they cause death. Therefore, these foods and products from animals represent a public health concern all over the world (Manthoor and Saliem, 2023; saad et al., 2024).

Highly dangerous pathogens such as E. coli O157:H7, Salmonella spp., Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus can be transmitted through eating meat and meat products (Abdulridha and Saliem, 2023; Al-Jaghifi and Khudhir, 2024a). Pathogenic bacteria with resistance to different kinds of antibiotics are gradually increasing, due to frequent and random use of antibiotics for veterinary treatments in attempts to prevent outbreaks of pathogens and to reduce the development of drug resistance among harmful microorganisms (Al-Jaghifi and Khudhir, 2024b). E. coli in animal-based foods is used as a contamination indicator of fecal materials and quality control practices, which include food production, food preparation, processing, handling, and market storage. Hence, the presence of coliform and E. coli is an indicator of food contamination by another pathogenic microorganism, such as Salmonella spp. and enteropathogenic bacteria, among others, which often threaten the safety of food. This is because these harmful bacteria are a serious public health concern. E. coli O157:H7 can be transmitted through the fecal-oral route or unhygienic practices for meat, during dressing livestock for storage and marketing (Dakheel et al., 2023). Domestic ruminants, which include cattle, goats, and sheep, are considered to be reservoirs of E. coli O157:H7 (Khudhir and Hammad, 2016). Consequently, meat and meat products from these livestock are often contaminated by E. coli O157:H7 (Khudhir and Hammad, 2016). Therefore, the objective of this study was to (i) confirm the prevalence and distribution of E. coli O157:H7 in beef sold in Baghdad city and (ii) examine the resistance statuses of E. coli and E. coli O157:H7 isolates from beef samples to some selected antibiotic drugs.

MATERIALS AND METHODS

Study Site and Sampling

This study was done from February 2023 to March 2024 in two regions located in Baghdad city (Al-Karkh and Al-Rusafa) of Iraq. A total of 140 beef samples were collected randomly: seventy (70) samples were obtained from retail butchers’ shops in each of the two regions in Baghdad city. Minced meat samples were transported in iceboxes immediately after collection. They were taken to the post-graduate milk and meat laboratory, Veterinary section of the Public Health Department, University of Baghdad, Iraq. Bacteriological tests were conducted within 2 hours after samples were collected on the same day.

Beef Samples Preparation for Bacteriological Analysis

One (1) kilogram of boneless beef samples was purchased from the local butcher shops in Baghdad city. These shops slaughtered the beef every morning to ensure freshness. The aim was to isolate E. coli O157:H7 using laboratory methods. Twenty-five grams of the beef was homogenized after adding 225 ml of modified tryptic soy broth (mTSB, Himedia, India) in the sterile polyethylene bags in the stomacher for 1-3 minutes. The mixture was incubated aerobically at 37 °C for 24 h (Haramain and Yagoub, 2021). After that, the culture was grown on MacConkey agar (MAC, Oxoid, UK) and eosin methylene blue agar (EMB, Oxoid, UK), incubated at 37 °C for 24 hours (Khudhir and Hammad, 2016), to get the visible colonies of bacteria. The isolates were confirmed as E. coli O157:H7 by plating onto the cefixime tellurite sorbitol MacConkey agar (CT-SMAC, Oxoid, UK), and HiCrome O157:H7 agar (HEC, Hi-Media, India) following the procedures of Shahid and Yousif, (2022). Isolates were examined using Gram staining, motility test, and biochemical tests (indole, methyl red, Voges-Proskauer, oxidase, and catalase) as shown in Table 1.

 

Table ١: Cultural and biochemical characterization of E. coli O157:H7.

Cultural	Cultural test	Characteristics
	MAC agar	Pink colonies
	EMB agar	Dark colonies with center and a greenish metallic sheen
	SMAC- CT agar	Colorless colonies
	Chromogenic agar	Mauve colonies
Biochemical	Biochemical tests	Result	Characteristics
	Gram Stain	Negative	Pink
	Motility Test	Positive	Motile
	Oxidase Test	Negative	light pink
	Indole Test	Positive	Red ring
	Catalase Test	Positive	Bubble formation
	Methyl Red Test	Positive	Red colure
	Voges Proskauer Test	Negative	Yellow colure

 

DNA Extraction

DNA extraction from bacterial cells was carried out by the G-spin Total DNA Extraction Kit, according to the manufacturer’s instructions. One or two mL of each culture was harvested by centrifugation (13000 rpm, 15 min, 65 °C). The sedimented colonies were washed five times, using phosphate buffered saline, and then suspended in 1.0 mL of sterilized water; they were kept at 4°C for the next step (Keeratikunakorn et al., 2024).

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) was performed using genomic DNA extracted from the isolated bacterial strains. A volume of 1 μL of DNA (approximately 200 ng) was added to each PCR reaction mixture. The thermal cycling conditions were as follows: an initial denaturation step at 94°C for 5 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute. A final extension step was carried out at 72°C for 5 minutes to complete the amplification process.

Primers

Primers used in this study were illustrated in Table 2, along with their sequences and concentrations.A total reaction volume was adjusted to 50 μl using deionized water. To prevent evaporation during the reaction, 40 μl of paraffin oil was added.

 

Table ٢: Primers used for detection of E. coli O157:H7.

Primer sequence 5'-3'	Genes	size of products	References
F-AAGATTGCGCTGAAGCCTTTG R-CATTGGCATCGTGTGGACAG	rfbE	497 bp	(Desmarchelier et al., 1998)
F-GCGCTGTCGAGTTCTATCGAGC R-CAACGGTGACTTTATCGCCATTCC	fliCH7	625 bp	Gannon et al., 199٧
F-CAGGTCGTCGTGTCTGCTAAA R-TCAGCGTGGTTGGATCAACCT	eaeA	1089 bp	Gannon et al., 199٣

 

Agarose Gel Electrophoresis

The amplified PCR products were analyzed by agarose gel electrophoresis, following the procedure described by Sambrook et al. (1989). A standard 100 bp DNA ladder was used as a molecular size marker to estimate the fragment sizes of the amplified products.

Antimicrobial Sensitivity Test

Confirmed Escherichia coli O157:H7 isolates were subjected to antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method on Mueller-Hinton Agar (MHA; Merck, Germany), following the Clinical and Laboratory Standards Institute (CLSI, 2020) guidelines. A total of 20 commercially available antibiotic discs (Oxoid, UK) were used: Amikacin (30 μg), Amoxicillin/Clavulanic Acid (30 μg), Ampicillin (10 μg), Cefixime (5 μg), Cefotaxime (30 μg), Cefoxitin (30 μg), Chloramphenicol (10 μg), Ciprofloxacin (5 μg), Colistin (25 μg), Doxycycline (30 μg), Gentamicin (10 μg), Imipenem (10 μg), Kanamycin (30 μg), Levofloxacin (5 μg), Nalidixic Acid (30 μg), Nitrofurantoin (300 μg), Ofloxacin (10 μg), Rifampicin (5 μg), Streptomycin (10 μg), and Tetracycline (10 μg).

Bacterial isolates were first cultured on nutrient agar and incubated at 37 °C for 24 hours. Following incubation, colonies were suspended in 5 mL of sterile 0.85% saline to achieve turbidity equivalent to 0.5 McFarland standard. Sterile cotton swabs were dipped into the standardized suspension, excess fluid was removed by pressing the swab against the inner wall of the test tube, and the inoculum was evenly spread over the surface of MHA plates.

Antibiotic discs were then placed on the inoculated plates using sterile forceps, ensuring a minimum spacing of 24 mm between discs and 10 mm from the plate edge. Plates were left at room temperature for 3–5 minutes to allow absorption and then incubated at 37 °C for 24 hours. Post-incubation, the diameters of the zones of inhibition were measured in millimeters using a digital caliper, and results were interpreted as susceptible, intermediate, or resistant according to CLSI standards.

The multiple antibiotic resistance (MAR) index for each isolate was calculated following the method described by Krumperman (1983), using the formula MAR = a/b, where a is the number of antibiotics to which the isolate was resistant, and b is the total number of antibiotics tested.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, Inc., USA). The Chi-square test for equal proportions was employed to assess whether the frequency of quality control bacterial isolates or E. coli O157:H7 strains varied significantly across different categorical groups of beef samples. A p-value of <0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Isolation and Identification of E. coli O157:H7 from Raw Beef Samples

The E. coli O157:H7 was isolated and identified from beef in Baghdad province using biochemical tests, simple media and selective media. On MacConkey agar, the colony appeared pink and non-mucoid due to the lactose fermentation. On EMB agar, the colonies exhibited a green metallic sheen, which aided in the preliminary identification of organism. These isolates were confirmed as E. coli by biochemical tests. The identified E. coli colonies were further sub-cultured onto CT-SMAC agar and HiCrome agar, followed by incubation at 37 °C for 24 hours. On CT-SMAC agar, E. coli O157:H7 colonies appeared colorless due to their inability to ferment sorbitol, a characteristic that distinguishes them from other E. coli serotypes. On HiCrome agar, the E. coli O157:H7 colonies exhibited a mauve to pink coloration. These phenotypic traits are consistent with the findings reported by Jabur et al. (2016).

Escherichia coli O157:H7 is a highly pathogenic bacterium in humans, and its detection and distribution are best confirmed through molecular characterization using specific virulence genes. In this study, a total of 140 randomly collected beef samples were analyzed to determine the prevalence and regional distribution of E. coli O157:H7 in Baghdad. The results showed that in the Al-Karkh region, 31 isolates of E. coli were identified, representing 44.29% of the contaminated samples. In the Al-Rusafa region, 27 isolates were detected, accounting for 38.57% of the contaminated samples. These findings highlight the widespread presence of E. coli in retail beef and underscore the importance of regional monitoring and molecular diagnostics for food safety surveillance.

The isolation process focused on the Karkh and Rusafa districts of Baghdad. E. coli O157:H7 was not detected in any of the samples collected from the Karkh region, as shown in Table 3. In contrast, 2 out of 70 samples (2.86%) from the Al-Rusafa region tested positive for E. coli O157:H7, as presented in Table 4. These findings align with previous studies conducted in Baghdad, where E. coli O157:H7 was also isolated from raw beef products, such as minced beef and beef burgers (Al-Obaidi and Dawood, 2023; Al-Jaradat et al., 2024b). Overall, the contamination rate of E. coli O157:H7 in this study was 2 out of 140 samples, equivalent to 1.42%.

 

Table ٣: Distribution of Coliform, E.coli and E.coli O157:H7 in beef samples collected from Al-Karkh region.

Districts	No. of samples	Coliform positive isolates (%)	E. coli positive isolates (%)	E. coli O157: H7 positive isolates
Abu Ghraib	10	5 (٥٠%)	5 (٥٠%)	0 (0%)
Al-A`amiriya	10	7 (٧٠%)	3 (٣٠%)	0 (0%)
Al-shu`ala	10	6 (٦٠%)	4 (٤٠%)	0 (0%)
Bayaa	10	3 (٣٠%)	7 (٧٠%)	0 (0%)
Dora	10	3 (٣٠%)	7 (٧٠%)	0 (0%)
Kadhimiya	10	8 (٨٠%)	2 (٢٠%)	0 (0%)
Seaidya	10	7 (٧٠%)	3 (٣٠%)	0 (0%)
Total	70	39 (55.71%)	31 (44.29%)	0 (0%)
Chi-square value		9.61	9.61	--
P-value		0.14 NS	0.14 NS	--

 

Table ٤: Distribution of Coliform, E.coli and E.coli O157:H7 in beef samples collected from Al-Rusafa region.

Districts	No. of samples	Coliform	E. coli	E. coli O157:H7
		Number of positive isolates (%)	Number of positive isolates (%)	Number of positive isolates(%)
Adhamiyah	10	7 (٧٠%)	3 (٣٠%)	0 (0%)
Bab Al-Moatham	10	8 (٨٠%)	2 (2٠%)	0 (0%)
Baghdad Al-Jadida	10	5 (٥٠%)	5 (٥٠%)	0 (0%)
Karrada	10	8 (٨٠%)	2 (2٠%)	0 (0%)
Sadr City	10	3 (٣٠%)	6 (٦٠%)	1 (١٠%)
Sha`ab	10	5 (٥٠%)	4 (٤٠%)	1 (١٠%)
Zayonua	10	5 (٥٠%)	5 (٥٠%)	0 (0%)
Total	70	41(58.57%)	27(38.57%)	2 (2.86%)
Chi-square value		8.59	3.15	1.15
P-value		0.16 NS	0.78 NS	0.97 NS

 

The two E. coli O157:H7 isolates were confirmed using conventional polymerase chain reaction (PCR) by detecting the presence of key virulence genes (eaeA, rfbE, and fliCH7), as shown in Figures 1 and 2. These findings are consistent with previous studies conducted both in Iraq and internationally. For instance, a study in Erbil, Iraq, reported that 3 out of 100 meat samples (6%) were positive for E. coli O157:H7 (Jalal and Aziz, 2023). Similarly, in Riyadh, Saudi Arabia, Hessain et al. (2015) found that 2% of 370 beef samples tested positive for the pathogen.

 

 

 

Although the overall contamination rate of E. coli O157:H7 in the present study was relatively low (1.42%), its presence in fresh beef sold at local butcher shops in Baghdad is concerning. This suggests potential lapses in food hygiene and handling practices that could pose risks to public health. Foods of animal origin, especially raw or undercooked meat, remain the primary sources of enterohemorrhagic E. coli (EHEC) infections. Contamination of meat products with this virulent strain underscores the need for improved meat inspection, handling, and consumer education to mitigate the risk of foodborne illness.

Beef carcasses can be significantly affected during the slaughterhouse processes when they come into contact that are contacted with infected workers or equipment under poor hygienic conditions in slaughterhouses. The exposure often leads to a contamination process by high initial bacterial loads, which ultimately have a strong impact on the hygiene characteristics of fresh beef that is sold in the Iraqi local markets. The isolation and distribution rate of both E. coli and E. coli O157:H7 between the two regions of Baghdad city may vary due to differences in hygienic practices. Additionally, contamination can occur during the handling of beef in the abattoir, especially if proper cleanliness is not maintained. Ensuring the cleanliness of slaughtering equipment and all subsequent steps leading to consumption is critical to minimizing risks.

In the present study, the E. coli O157:H7 isolates demonstrated multidrug-resistant (MDR) properties, as illustrated in Figure 3. The pathogenicity of these isolates was further confirmed by the detection of virulence genes (eaeA, rfbE, and fliCH7), as shown in Table 5. The presence of genes associated with both virulence and antibiotic resistance raises serious public health concerns, particularly as these resistance determinants can be transmitted to consumers through contaminated food and improper handling practices (Rahman et al., 2017).

 

Table 5: Number and percentage (%) of eaeA, rfbE, and fliCH7 virulence genes detected in E. coli O157:H7 isolates.

Source	Positive isolates of E. coli O157:H7	eaeA	rfbE	fliCH7	%
Beef samples	2	2	2	2	100
Total	2	2	2	2	100

 

The misuse and unregulated application of antibiotics in livestock farming are significant contributing factors to the emergence of MDR pathogens. Commonly used antibiotics in cattle rearing include tetracyclines, penicillins, and sulfonamides. These are frequently administered under poor hygienic conditions, especially in slaughterhouses and during meat processing, increasing the risk of contamination. Khudhir (2020) noted that although various processing methods can significantly reduce the presence of E. coli O157:H7 in meat, these reductions may not be sufficient to guarantee food safety. Cross-contamination and inadequate hygiene practices during processing remain critical points of concern (Fayemi et al., 2021).

Beta-lactam antibiotics such as penicillin, tetracyclines like tetracycline, and aminoglycosides such as erythromycin, along with trimethoprim/sulfamethoxazole, are widely used in both human and veterinary medicine (Faghihi et al., 2017; Mirza, 2015). Interestingly, some E. coli O157:H7 strains in this study exhibited susceptibility to certain antimicrobial agents still employed in the veterinary field, a finding consistent with previous studies (Ayenew et al., 2021).

Antibiotic-resistant zoonotic E. coli pose significant challenges to public health and are associated with increased hospitalization costs. In severe cases, particularly among immunocompromised individuals such as children and the elderly, these infections may be fatal (Onwumere-Idolor et al., 2024). The E. coli O157:H7 isolates in this study exhibited resistance to multiple antibiotics, including sulfonamides, streptomycin, tetracycline, and ampicillin, consistent with previous findings (Guzmán et al., 2019).

These results underscore the urgent need to address the threat posed by antibiotic-resistant E. coli O157:H7, especially in the context of widespread antibiotic use for therapeutic and growth-promoting purposes in livestock. The MAR index was used to trace the origin of bacterial contamination (Adzitey, 2015; (Davis and Brown, 2016). A MAR index greater than 0.2 suggests that the isolates originated from environments where antibiotics are frequently used or misused (Krumperman, 1983; Mthembu et al., 2019).

Consistent with findings by Abebe et al. (2023), the E. coli O157:H7 isolates in the current study exhibited resistance to more than three antimicrobial agents. Specifically, 56% of the isolates were resistant to 4 antibiotics, while 44% demonstrated resistance to five, highlighting the severity of antibiotic resistance in foodborne pathogens.

CONCLUSIONS AND RECOMMENDATIONS

The results of this study indicated that the prevalence of antibiotic-resistant E. coli O157:H7 in local beef samples from Baghdad was relatively low. Nevertheless, the detection of even a small number of contaminated samples is a significant concern and highlights a potential threat to food safety and public health. The low isolation rate observed may be attributed to increased consumer awareness of hygiene and health practices following the COVID-19 pandemic, along with improved adherence to food safety protocols during meat handling. Additionally, stricter health monitoring and regulation of butcher shops likely played a role in minimizing the spread of these resistant bacterial strains.

ACKNOWLEDGEMENTS

Thanks, the head of high graduated milk and meat Laboratory, university of Baghdad for provided all assists for complete the current research.

NOVELTY STATEMENT

The novelty of this research lies in its focus on determining the distribution and percentage of antibiotic-resistant Escherichia coli O157:H7 isolated from locally sold beef in Iraqi markets. This study also evaluates the hygiene standards of local meat by comparing bacterial contamination loads across different regions of Baghdad. Such a regional assessment provides valuable insight into public health risks associated with foodborne pathogens and contributes to the understanding of antimicrobial resistance trends in Iraq.

AUTHOR’S CONTRIBUTIONS

Ibraheem Eibrhim take the responsibility of data collection, bacteriological isolation, preparing the manuscript and data analysis while research design due the supervision of advisor Dr Zina Saab Khudhir. Both the authors approved the final version of the manuscript that submitted to this respect journal.

Conflict of Interest

No potential conflicts of interest relevant to this article are reported.

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