Molecular Characterization of Antimicrobial Resistance Genes in Processed Red Meat-Derived Escherichia coli Isolates

 Corresponding author: Muhsin AYDIN Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Molecular Characterization of Antimicrobial Resistance Genes in Processed Red MeatDerived Escherichia coli Isolates Seher SAYGI 1 and Muhsin AYDIN 2, * 1 Department of Biology, Graduate Education Institute, Adıyaman University, Adıyaman, 02040, Türkiye. 2 Department of Biology, Faculty of Arts and Science, Adıyaman University, Adıyaman, 02040, Türkiye. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 Publication history: Received on 19 September 2025; revised on 25 October 2025; accepted on 27 October 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.33.1.0416 Abstract This study aimed to determine the presence of extended-spectrum β-lactamase (ESBL) genes (blade, blush, bloat, blastM), sulfonamide resistance genes (sul1, sul2, and sul3), and integrin genes (int1, int2, and int3) in Escherichia coli isolates isolated from processed red meat samples collected in Adıyaman Province, Türkiye. A total of 14 E. coli isolates were analyzed using a molecular technique. PCR results revealed that several isolates carried multiple resistance genes simultaneously. Isolates harboring both ESBL and sulfonamide resistance genes were identified as 19B, 16B, 15B, and 2B, while those carrying integrin and sulfonamide resistance genes were 14A, 19A, 19B, 13A, and 15B. Only isolates 19B and 15B contained all three gene groups. These findings indicate that foodborne E. coli strains may possess multiple antibiotic resistance mechanisms, posing a potential public health risk. The study highlights the importance of implementing molecular surveillance programs to monitor antibiotic resistance within the food safety framework. Keywords: Escherichia coli; Antibiotic Resistance; ESBL; Sulfonamide; Integrin 1. Introduction The widespread and uncontrolled use of antibiotics has accelerated the dissemination of resistance genes among bacteria, leading to a serious global public health issue [1]. Antimicrobial resistance (AMR) is no longer confined to hospital environments but has spread across the environment, livestock production, and the food chain [2]. The use of antibiotics as prophylactic or growth-promoting agents in animal husbandry contributes to the emergence of resistant bacterial strains. These bacteria can be transmitted to humans via food and cause infections that are difficult to treat [3]. Escherichia coli is a commensal bacterium naturally found in the intestinal flora of humans and animals. However, certain E. coli strains have acquired genetic determinants that enable them to resist multiple classes of antibiotics. Extended-spectrum β-lactamases (ESBLs) hydrolyze β-lactam antibiotics, such as cephalosporins and monobactams, thereby reducing the effectiveness of treatment. Consequently, ESBL-producing E. coli strains have become important pathogens in both community-acquired and foodborne infections [4]. Resistance to sulfonamide antibiotics is primarily mediated by sul genes (sul1, sul2, and sul3), which are often located on mobile genetic elements such as plasmids and transposons, allowing horizontal gene transfer among bacterial populations [5]. Similarly, integrins are genetic structures capable of capturing and transferring resistance gene cassettes, playing a major role in the dissemination of antibiotic resistance genes [6]. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 301 Recent studies have reported the simultaneous presence of multiple resistance genes in foodborne E. coli isolates. This co-occurrence increases the potential transmission of resistant bacteria through the food chain, posing a significant threat to public health [7]. The aim of this study was to investigate the presence and co-occurrence of ESBL, sulfonamide resistance, and integron genes in E. coli isolates obtained from processed red meat samples in Adıyaman Province, Türkiye. The findings of this research will contribute to the understanding of antibiotic resistance mechanisms in foodborne bacteria and support regional antimicrobial resistance monitoring efforts. 2. Materials and Methods 2.1. Samples and Isolation of E. coli Strains In this study, genotypic identification and molecular characterization of Escherichia coli strains isolated from processed red meat samples were conducted. A total of 14 red meat samples were analyzed. All samples were aseptically collected in sterile polyethylene bags, transported under cold-chain conditions, and processed within 2–4 hours of collection to minimize bacterial overgrowth. For bacterial isolation, 25 g of each meat sample was homogenized in 225 mL of phosphate-buffered saline (PBS, pH 7.6) using a stomacher homogenizer. Aliquots of 100 µL from appropriate dilutions were plated onto MacConkey agar and Eosin Methylene Blue (EMB) agar media. The inoculated plates were incubated at 37°C for 18–24 hours under aerobic conditions. After incubation, colonies with typical E. coli morphology were identified based on their characteristic appearance—pink to red colonies on MacConkey agar and metallic green sheen colonies on EMB agar. Representative colonies were repeatedly sub cultured to obtain pure isolates, which were preserved at −20°C for molecular analyses. 2.2. DNA Extraction Genomic DNA was extracted from the confirmed E. coli isolates using the boiling method. Briefly, bacterial colonies were suspended in nuclease-free water, heated at 95°C for 10 minutes, and centrifuged. The supernatant containing DNA was collected and stored at −20°C until further use as a PCR template. 2.3. Molecular Confirmation of E. coli (uspA Gene) Molecular confirmation of phenotypically identified isolates was performed by polymerase chain reaction (PCR) as suggested by Chen and Griffiths (1998) [8] targeting the spa gene, which encodes a universal stress protein specific to E. coli. The reference strain E. coli ATCC 25922 was used as a positive control. PCR conditions were adapted from the method described by Chen and Griffiths (1998) and optimized for the present study. Reaction mixtures and thermal cycling parameters followed a standard PCR protocol. PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized under a UV transilluminator. Samples yielding an amplicon of 884 bp were considered molecularly confirmed as E. coli. 2.4. PCR Analysis of Resistance Genes Ten target genes were investigated by polymerase chain reaction (PCR), including ESBL genes (blaCTX-M, blaSHV, blaOXA, and blaTEM), sulfonamide resistance genes (sul1, sul2, and sul3), and integron class genes (int1, int2, and int3). PCR reactions were performed in a total volume of 25 µL, and the specific thermal cycling conditions for each gene are summarized in Table 3.4. Each reaction mixture contained Master Mix, forward and reverse primers, template DNA, and nuclease-free water. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 302 Table 1 PCR amplification conditions and references for target genes used in E. coli isolates Target Gene Initial Denaturation (°C/min) Denaturation (°C/s) Annealing (°C/s) Extension (°C/s) No. of Cycles Final Extension (°C/min) Reference blaTEM 94 / 5 94 / 30 55 / 30 72 / 45 35 72 / 7 [9] blaSHV 94 / 5 94 / 30 56 / 30 72 / 45 35 72 / 7 blaOXA 94 / 5 94 / 30 52 / 30 72 / 45 35 72 / 7 blaCTX-M 94 / 5 94 / 30 60 / 30 72 / 45 35 72 / 7 sul1 94 / 5 94 / 30 58 / 30 72 / 45 35 72 / 7 [10] sul2 94 / 5 94 / 30 55 / 30 72 / 45 35 72 / 7 sul3 94 / 5 94 / 30 54 / 30 72 / 45 35 72 / 7 int1 94 / 5 94 / 30 56 / 30 72 / 45 35 72 / 7 [11] int2 94 / 5 94 / 30 57 / 30 72 / 45 35 72 / 7 int3 94 / 5 94 / 30 58 / 30 72 / 45 35 72 / 7 All PCR reactions were performed in 25 µL volumes using gene-specific primers under the conditions described above. 2.5. Agarose Gel Electrophoresis PCR products were separated on a 1.5% agarose gel prepared in 1X TBE buffer, stained with ethidium bromide, and visualized using a Viler Lurma UV gel documentation system. A 100 bp DNA ladder was used as a molecular marker. 2.6. Statistical Analysis The data were analyzed descriptively, and gene distribution frequencies were expressed as percentages. 3. Results and Discussion 3.1. Detection of ESBL Genes The molecular analysis of extended-spectrum β-lactamase (ESBL) gene variants in E. coli isolates obtained from processed red meat samples demonstrated a limited distribution of β-lactamase genes. Among the 14 isolates analyzed, the blaCTX-M gene was detected in only one isolate (15B), whereas the bloat gene was identified in five isolates (15A, 19B, 21A, 16B, and 2B). No amplification was observed for the blaTEM and blaSHV genes. The overall distribution of the ESBL genes detected in E. coli isolates is summarized in Table 2. Representative agarose gel electrophoresis images showing PCR amplification of the blaOXA and blaCTX-M genes are presented in Figure 1. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 303 Table 2 Distribution of ESBL genes in E. coli isolates Number Isolate Code blaCTX-M blaOXA blaSHV blaTEM 1 18 - - - - 2 13B - - - - 3 15A - + - - 4 14A - - - - 5 19A - - - - 6 19B - + - - 7 9B - - - - 8 13A - - - - 9 21A - + - - 10 12B - - - - 11 19D - - - - 12 16B - + - - 13 15B + - - - 14 2B - + - - Figure 1 Agarose gel electrophoresis image showing PCR products of blaOXA, blaTEM, and blaCTX-M genes The detection of blaOXA in five isolates and blaCTX-M in a single isolate indicates a relatively low prevalence of ESBLproducing E. coli strains in the analyzed meat samples. The absence of blaTEM and blaSHV genes suggests that these older ESBL variants may be gradually replaced by newer β-lactamase types such as blaCTX-M and blaOXA in this geographical region. When compared with data from other countries, notable differences can be observed. In India, blaCTX-M and blaTEM were predominant among ESBL-positive E. coli strains isolated from street foods, detected in 69.04% and 66.66% of isolates, respectively, while blaOXA was found in 19.04% and blaSHV was absent [12]. In Germany, 10.1% of red meat isolates carried blaCTX-M, whereas blaSHV and blaTEM were detected at lower frequencies (2.0% and 0.8%, respectively) [13]. Similarly, studies in Taiwan reported blaCTX-M as the dominant ESBL gene among multidrug-resistant E. coli strains from bovine carcasses [14], whereas in Mexico, blaCTX-M was detected in 20% of retail meat isolates, with no detection of blaTEM, blaSHV, or blaOXA [15]. In addition, a study conducted in the northern regions of Egypt reported that among E. coli isolates obtained from raw beef and mutton, blaTEM, blaCTX-M, and blaOXA genes were detected in 52.4%, 42.9%, and 14.3% of isolates, respectively [16]. Compared with these results, the present study showed a markedly lower prevalence of ESBL genes, suggesting geographical variations in antibiotic usage and resistance dissemination patterns across neighboring Mediterranean region. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 304 The predominance of blaOXA over blaCTX-M in the present study may be related to regional differences in antibiotic usage practices, livestock management, and environmental selection pressures in Türkiye. The relatively low overall ESBL detection rate may also reflect limited use of third-generation cephalosporins in local animal farming or differences in sample type and size. The identification of blaOXA and blaCTX-M positive E. coli isolates, even at low frequency, is of particular concern, as these genes are often plasmid-mediated and can spread rapidly among bacterial populations via horizontal gene transfer [17]. These findings highlight the need for continuous surveillance of antimicrobial resistance in foodborne bacteria and emphasize the importance of prudent antibiotic use in livestock production to minimize the risk of dissemination to humans through the food chain. 3.2. Detection and Discussion of Sulfonamide Resistance Genes The molecular screening of sulfonamide resistance genes (sul1, sul2, and sul3) among E. coli isolates obtained from processed red meat samples revealed variable gene distributions. As shown in Table 3, the sul1 gene was detected in isolates 19A, 13A, and 12B; sul2 was identified in isolates 18, 13B, 14A, 19A, 19B, 13A, 12B, 16B, and 2B; and sul3 was detected in isolates 18, 14A, and 15B. Representative agarose gel electrophoresis images of sul1/sul2 and sul3 amplification products are presented in Figures 2 and 3, respectively. Table 3 Distribution of sul Genes in E. coli Isolates Number Isolate Code sul1 sul2 sul3 1 18 - + + 2 13B - + - 3 15A - - - 4 14A - + + 5 19A + + - 6 19B - + - 7 9B - - - 8 13A + + - 9 21A - - - 10 12B + + - 11 19D - - - 12 16B - + - 13 15B - - + 14 2B - + - GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 305 Figure 2 Agarose gel electrophoresis showing amplification of sul1 and sul2 genes in E. coli isolates Figure 3 Agarose gel electrophoresis showing amplification of sul3 gene in E. coli isolates Multiple gene combinations were observed in several isolates. Specifically, sul1 and sul2 coexisted in isolates 13A, 19A, and 12B, while sul2 and sul3 were simultaneously detected in isolates 18 and 14A. These results indicate that some E. coli strains may carry multiple sulfonamide resistance determinants, potentially enhancing their ability to survive under selective antibiotic pressure. When compared with results reported from other countries, noticeable variations in sul gene distribution patterns can be observed. In Spain, Ramos et al. reported that among E. coli strains isolated from pork meat, 23.1% harbored both sul1 and sul2 genes, 3.1% carried sul2 and sul3, and 1.5% carried sul1 and sul3 together; notably, only one isolate contained all three sul genes simultaneously [18]. In Canada, a study investigating E. coli isolates from a commercial beef processing plant found that sul1 was present in samples from hides (6%), washed carcasses (20%), conveyor belts (7%), beef trimmings (15%), and ground beef (4%), whereas sul2 was detected in 9%, 10%, 12%, 4%, and 15% of these respective sample types, and no isolates carried sul3 [19]. Similarly, an Austrian study analyzing E. coli from multiple meat sources (pork, beef, poultry, and minced meat) found that among 142 sulfonamide-resistant isolates, sul2 (n = 113) was far more prevalent than sul1 (n = 32), with only three isolates carrying both genes [20]. The findings of the present study are consistent with these international reports, as sul2 was also the most frequently detected gene, followed by sul1 and sul3. The detection of multiple sul genes in individual isolates suggests the potential for horizontal gene transfer and co-selection mechanisms under antibiotic pressure. The relatively lower detection rate of sul3 compared with sul1 and sul2 aligns with previous observations that sul3 is a less common variant, often restricted to specific plasmid types or ecological niches. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 306 Overall, these results highlight the persistent presence of sulfonamide resistance determinants in foodborne E. coli and underscore the necessity of ongoing monitoring to assess their role in the broader dissemination of antimicrobial resistance within the food chain. 3.3. Detection and Discussion of Integrin Genes The molecular screening of E. coli isolates obtained from processed red meat samples revealed the presence of the int1 gene, whereas int2 and int3 were not detected in any of the samples. As shown in Table 4, the int1 gene was identified in isolates 18, 14A, 19A, 19B, 13A, and 15B. Representative agarose gel electrophoresis images showing PCR amplification of the int1 gene are presented in Figure 4. Table 4 Distribution of int1, int2, and int3 genes among E. coli isolates obtained from processed red meat samples Number Isolate Code int1 int 2 int 3 1 18 + - - 2 13B - - - 3 15A - - - 4 14A + - - 5 19A + - - 6 19B + - - 7 9B - - - 8 13A + - - 9 21A - - - 10 12B - - - 11 19D - - - 12 16B - - - 13 15B + - - 14 2B - - - Figure 4 Agarose gel electrophoresis showing amplification of the int1 gene in E. coli isolates GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 307 The detection of int1 in 6 of the 14 isolates indicates that class 1 integrons are present among E. coli strains isolated from processed meat products, while the absence of int2 and int3 genes suggests limited dissemination of these integron classes in the analyzed samples. Integrons play a key role in the horizontal transfer of antibiotic resistance genes, and the identification of int1-positive isolates highlights their potential role in the spread of multidrug resistance determinants. Similar observations have been reported in previous studies conducted in different geographical regions. In Norway, class 1 and class 2 integrons were detected in 12% (int1) and 6% (int2) of 241 resistant E. coli strains isolated from meat products, respectively [21]. In contrast, a study conducted in South Korea identified class 1 integrons (int1) in 16 isolates from animal-derived foods, while class 2 and class 3 integrons were not detected, consistent with the findings of the present study [22]. Likewise, in India, 56 of 96 (58.3%) multidrug-resistant E. coli isolates from ready-to-eat food samples were positive for class 1 integrons, showing a significant correlation between integron occurrence and food origin [23]. Taken together, these results indicate that the presence of class 1 integrons in E. coli isolates from meat products is a globally observed phenomenon, although their prevalence varies by country and sample type. The detection of int1 in this study, even at a moderate frequency, suggests that processed meat products may act as reservoirs for integronassociated resistance genes. Continuous monitoring of integron-bearing E. coli isolates is therefore essential to better understand their role in the dissemination of antimicrobial resistance through the food chain. 3.4. Overall Evaluation The combined evaluation of ESBL, sulfonamide, and integron gene profiles revealed a clear indication of multidrug resistance among E. coli isolates obtained from processed meat samples. Although the overall detection frequency of individual genes was relatively low, several isolates harbored multiple resistance determinants simultaneously, suggesting the presence of mobile genetic elements mediating horizontal gene transfer. The predominance of blaOXA and sul2 genes, together with int1-positive isolates, highlights the potential linkage between β-lactamase, sulfonamide, and integron-associated resistance mechanisms. Such co-localization increases the risk of multidrug resistance dissemination through the food chain. These findings underline the importance of integrated molecular surveillance strategies that simultaneously target ESBLs, sul genes, and integron classes to provide a more complete understanding of antimicrobial resistance dynamics in foodborne E. coli. 4. Conclusion This study provides a comprehensive molecular characterization of Escherichia coli isolates obtained from processed red meat samples, focusing on β-lactamase (ESBL), sulfonamide resistance, and integron genes. The detection of blaOXA and blaCTX-M genes, along with the widespread occurrence of sul2 and the presence of class 1 integrons (int1), indicates that foodborne E. coli strains may act as reservoirs of multiple resistance determinants. The coexistence of these genes in certain isolates suggests possible plasmid-mediated horizontal gene transfer events contributing to the dissemination of multidrug resistance within the food chain. Although the overall gene prevalence was moderate, these findings highlight the need for continuous molecular surveillance to monitor the spread of antimicrobial resistance. Strengthening hygiene standards, regulating antibiotic use in animal husbandry, and implementing integrated resistance monitoring systems are crucial steps to prevent further dissemination of resistant E. coli from food sources to humans. Compliance with ethical standards Acknowledgements This study was supported by The Scientific and Technological Research Council of Türkiye (TÜBİTAK) 2209-A program, Project No: 1919B012401787. Disclosure of conflict of interest No conflict of interest to be disclosed. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 300-309 308 References [1] Muteeb G, Rehman MT, Shahwan M, Aatif M. Origin of Antibiotics and Antibiotic Resistance, and Their Impacts on Drug Development: A Narrative Review. Pharmaceuticals. 2023; 16(11):1615. [2] Ahmed SK, Hussein S, Qurbani K, Ibrahim RH, Fareeq A, Mahmood KA, et al. Antimicrobial resistance: Impacts, challenges, and future prospects. J Med Surg Public Health. 2024; 2:100081. [3] Manyi-Loh C, Mamphweli S, Meyer E, Okoh A. Antibiotic Use in Agriculture and Its Consequential Resistance in Environmental Sources: Potential Public Health Implications. Mol J Synth Chem Nat Prod Chem. 2018; 23(4):795. 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