The Prevalence of Antibiotic-Resistant Genes in Escherichia coli Isolated from the Intestines of Imported Sheep

1. Introduction

The normal flora (or microbiota) in animals consists of a diverse community of microorganisms, including bacteria, fungi, viruses, and protozoa, that naturally inhabit various body surfaces such as the skin, gastrointestinal tract, respiratory tract, and urogenital system. These microorganisms play essential roles in health, digestion, and immunity. Naturally occurring microorganisms, a diverse group of microorganisms that naturally inhabit animals, play a significant role in maintaining animal health and assisting in digestion processes [1]. In animals such as sheep, these bacteria are found in various parts of the body, particularly in the digestive system (intestines), skin, and mucous membranes. They include Lactobacillus spp., Bacteroides spp., Clostridium spp., Escherichia coli (commensal strains), and Ruminococcus spp. Sheep (Ovis aries) are one of the oldest domesticated animals and serve multiple purposes in agriculture, industry, and scientific research. Their versatility makes them valuable in various sectors, including food production, the fiber industry, and even medical research. Their meat is tender and widely consumed (lamb or mutton), more flavorful, and commonly eaten in many parts of the world, including the Middle East, Asia, and Africa. Sheep hold great significance in Islamic culture, religion, and daily life. Sheep are deeply woven into the religious, economic, and cultural fabric of the Islamic world [2]. Their role in sacrificial rituals, food, trade, and traditional customs makes them one of the most valued livestock animals in Muslim communities. E. coli are among the most important natural bacteria present in sheep, forming part of the “microbiome” (the natural microbial community). Their role includes improving the health of sheep by aiding digestion, where bacteria in the intestines and rumen help break down fibers and starches, enhancing nutrient absorption efficiency and boosting sheep health [3]. They also enhance immunity, acting as a first line of defense against harmful organisms by competing for resources and space, and contribute to the production of vitamins that improve the overall health of sheep. Moreover, the balance between different bacteria helps prevent the spread of pathogenic bacteria [4]. The introduction of antimicrobial agents in animal husbandry, whether for treatment, prevention, or growth promotion, inadvertently contributed to the selection and persistence of antibiotic-resistant bacteria [5]. The overuse and misuse of antibiotics in livestock create a favorable environment for the development and spread of bacterial resistance genes. Consequently, these genes can be transferred between bacteria, promoting the emergence of multidrug-resistant strains, posing a significant public health threat, and potentially affecting sheep health and productivity. These genes make it difficult to treat infections with conventional antibiotics, necessitating the use of more effective antibiotics or combinations of several types [6]. Resistance of intestinal bacteria to antibiotics is a pressing concern in both animal and public health. The resistance of intestinal bacteria in sheep to antibiotics is a complex problem that requires serious attention from farmers and veterinarians. Managing the prudent use of antibiotics and adopting comprehensive health strategies can help reduce the spread of resistance and preserve the efficacy of antibiotics for the future [7].

The presence of antibiotic-resistance genes in gram-negative intestinal bacteria in sheep poses a significant challenge to both animal and human health [8]. These genes complicate the treatment of infections using conventional antibiotics, necessitating the use of more potent antibiotics or combinations of antibiotics. The diversity of antibiotic-resistance genes in intestinal bacteria indicates that these bacteria can resist a wide range of antibiotics, complicating treatment and increasing the risk of transferring these resistant bacteria to humans or other environments. Controlling the spread of these genes requires optimal antibiotic use in animal husbandry, alongside rigorous and continuous monitoring programs and preventive measures to avoid bacterial contamination in sold meat [9].

The spread of bacterial resistance genes in the intestines of animals is a major concern due to its potential impact on public health, as it can lead to the transfer of resistance to human pathogens [10]. Horizontal gene transfer of resistance genes between different bacterial species can result in the creation of more challenging strains. If resistant bacteria from meat enter the human body, they may cause infections that are difficult to treat with conventional antibiotics, significantly limiting treatment options for bacterial infections. The presence of resistance genes in the normal flora of sheep also has a major impact on the environment. Resistant bacteria from sheep can be introduced into soil, water, and wildlife, leading to the spread of antimicrobial resistance across ecosystems. Therefore, monitoring and detecting outbreaks of infectious diseases is crucial in protecting consumers and highlighting critical points during both production and consumption stages. Studying the prevalence of bacterial resistance genes in the intestines of animals is essential for protecting public health, ensuring food safety, and promoting the responsible use of antibiotics in both animal and human healthcare [11]. This chapter aimed to assess the prevalence of antibiotic-resistance genes in E. coli isolated from the intestines of imported sheep in the Western region of KSA.

2. Methodology

3. Results

Table 1 summarizes the key findings from the study. A total of 49 Gram-negative bacterial strains were isolated from the intestines of sheep. E. coli was the most frequently isolated species, accounting for 53.1% (26/49) of the total isolates. Other bacterial species included Enterobacter cloacae (30.6%), Klebsiella pneumoniae (4.1%), Salmonella paratyphi A (2%), Acinetobacter haemolyticus (2%), Klebsiella intermedia (2%), Enterobacter aerogenes (2%), Aeromonas caviae (2%), and Tatumella (2%) as shown in Figure 1. The antibiotic susceptibility testing of the E. coli isolates revealed varying resistance levels (Figure 2). High resistance was observed against ampicillin (88.5%), trimethoprim-sulfamethoxazole (80.8%), and cefuroxime (65.4%). Resistance to cefepime (61.5%) and levofloxacin (57.7%) was also significant. Notably, no resistance was detected against amoxicillin-clavulanate and piperacillin-tazobactam. ESBL production was identified in 50% (13/26) of the E. coli isolates. Molecular analysis of the antibiotic resistance genes in E. coli isolates revealed a high prevalence of sul2 (96.2%), followed by aadA1 (73.1%) and tet_A (65.4%), as shown in Figures 3 and 4. The sul1 and sul3 genes were detected in 46.2% and 42.3% of isolates, respectively. The blaSHV gene was present in 34.6%, while strA_strB was detected in 30.8%. Other genes, including tet_C (15.4%), cat (15.4%), blaTEM (3.8%), and qnr (0%), were found at lower frequencies. Table 2 shows the distribution of antibiotic resistance gene multiplicity in E. coli isolates, which reveals significant variability. Only one isolate (3.8%) carried no resistance genes, while two isolates (7.7%) carried two genes. Three and four gene combinations were observed in 19.2 and 7.7% of isolates, respectively. Notably, 50% of isolates carried five resistance genes, and 11.5% harbored six resistance genes, indicating a high level of genetic resistance diversity. Statistical analysis using Pearson’s chi-square test showed significant associations between ESBL production and resistance to specific antibiotics (Table 3). Resistance to cefepime (p = 0.000), cefazolin (p = 0.014), aztreonam (p = 0.020), and ceftazidime (p = 0.020) were significantly correlated with ESBL production. However, no significant association was found with resistance to cefotaxime (p = 0.269) and cefoxitin (p = 0.396).

4. Discussion

Sheep are among the most widely consumed types of meat in the world in addition to their nutritional benefits [22]. The gut microbiota of sheep naturally harbors resistant bacteria, which can spread within livestock populations and potentially transfer to humans. While normal flora plays a vital role in maintaining gut health and digestion, the spread of resistance genes can contribute to the emergence of antibiotic-resistant bacteria, which can be transferred to humans, animals, and ecosystems.

The findings of chapter demonstrate a substantial prevalence of E. coli among gram-negative bacterial isolates from sheep intestines. The dominance of E. coli(53.1%) is consistent with previous studies highlighting its widespread presence in livestock intestines [23, 24]. The significance of E. coli lies in the fact that some strains, such as E. coli O157, can be pathogenic and cause food poisoning if transmitted to humans [24]. The presence of other bacterial species, such as Enterobacter cloacae and Klebsiella pneumoniae, further indicates the diverse microbial community within the animal gut, some of which can act as opportunistic pathogens. These results were consistent with global studies confirming that E. coli is the most prevalent intestinal bacteria in sheep [25, 26]. The aforementioned percentages reflect the natural presence of bacteria in the intestines; however, some can pose a public health risk if they become pathogenic or resistant to antibiotics.

The high resistance rates observed in E. coli, particularly against ampicillin (88.5%) and trimethoprim-sulfamethoxazole (80.8%), raise significant concerns regarding the efficacy of commonly used antibiotics. The high resistance levels to these antibiotics are concerning because they are commonly used to treat a wide range of bacterial infections, and this resistance may affect both animal and public health. This was similar to a study conducted in Qatar, where the highest resistance in E. coli was to ciprofloxacin (69.4%) and trimethoprim/sulfamethoxazole (45.8%) [27]. A global study also indicated that 13 types of gram-negative bacteria isolated from goats and sheep intestines were resistant to various antibiotics, including penicillin, ampicillin, amoxicillin, chloramphenicol, and others [28]. Resistance to beta-lactam antibiotics, especially third- and fourth-generation cephalosporins like cefepime (61.5%) and cefuroxime (65.4%), suggests the emergence of extended-spectrum beta-lactamase (ESBL)-producing strains. These findings highlight the potential risk of antimicrobial resistance transmission from animals to humans through direct contact or food consumption. Thus, antibiotic resistance in these intestinal bacteria poses a complex challenge that requires serious attention from farmers and veterinarians, and comprehensive health strategies need to be adopted to reduce the spread of resistance and maintain the effectiveness of antibiotics in the future.

The molecular analysis further reinforces these concerns, as the widespread presence of resistance genes, such as sul2 (96.2%) and aadA1 (73.1%), indicates an extensive genetic basis for resistance. The presence of antibiotic-resistance genes in the normal flora of sheep is a growing concern with serious implications for animal health, human health, and environmental sustainability. The transmission of resistant bacteria through direct contact, food, and environmental contamination highlights the urgent need for better antibiotic stewardship, hygiene measures, and surveillance programs to reduce the impact of antimicrobial resistance. A similar study found an increase in resistance in bacteria isolated from livestock feces, with common genes such as sul3, cat, qnr, qnrB, and tet(A) [29]. The problem is that these genes enable bacteria to resist various antibiotics, complicating the treatment of related infections. Sulfonamide resistance genes (sul1, sul2, sul3) are widespread in bacteria and make them resistant to sulfonamides such as sulfamethoxazole [30]. Aminoglycoside-modifying enzymes (AMEs), such as aadA1, are among the most prevalent genes in intestinal bacteria, rendering antibiotics like gentamicin, tobramycin, and amikacin ineffective. Likewise, tetracycline resistance genes (tet(A), tet(B), tet(M)), beta-lactamase genes (SHV), and chloramphenicol resistance genes (cat) are also widespread [29, 31]. The presence of these resistance genes in gram-negative bacteria in the intestines of sheep and lambs presents a significant challenge to public health. These genes make it difficult to treat infections with traditional antibiotics, and controlling their spread requires strict policies on antibiotic use in livestock farming, along with continuous monitoring programs. The detection of ESBL-related genes (blaSHV in 34.6% and blaTEM in 3.8% of isolates) suggests that some strains have acquired mechanisms to hydrolyze a wide range of beta-lactam antibiotics. Notably, the absence of qnr genes suggests that resistance to fluoroquinolones may be mediated by alternative mechanisms.

The gene multiplicity analysis highlights a concerning trend, with half of the E. coli isolates carrying five resistance genes. This suggests that a significant proportion of the bacterial population possesses multiple resistance determinants, increasing the likelihood of multidrug resistance. The detection of isolates with up to six resistance genes further emphasizes the genetic adaptability of E. coli and its potential to evade multiple antibiotic classes. This high level of gene accumulation poses a serious threat to antimicrobial efficacy, as bacteria carrying multiple resistance determinants can spread resistance traits more effectively within microbial communities. The presence of multiple antibiotic-resistance genes complicates treatment and increases the risk of transmitting resistant bacteria to humans or other environments, especially due to the widespread use of antibiotics in sheep farming [32]. The health and environmental challenges posed by multiple resistance genes make bacterial infections harder to treat, and multi-resistant bacteria can spread easily between animals and humans through the food chain or direct contact. This could reduce the effectiveness of antibiotics in agriculture, leading to higher animal mortality rates and potential public health risks [33, 34]. Bacteria with multiple resistance genes can survive even with the use of multiple antibiotics, making treatment more difficult. Also, the spread of bacteria carrying multiple resistance genes poses a risk of transferring these genes to other bacteria, in the environment or other living organisms, including humans, through the food chain or direct contact. The treatment challenges also include the reduced availability of common antibiotics, requiring veterinarians to resort to less common or more expensive antibiotics, which may have greater side effects on animals [35].

The statistical correlation between ESBL production and resistance to third- and fourth-generation cephalosporins (cefepime, cefazolin, aztreonam, and ceftazidime) aligns with global reports on the increasing prevalence of ESBL-producing E. coli in both human and veterinary medicine. This finding emphasizes the need for improved surveillance and stricter antimicrobial usage policies in livestock production to mitigate the further spread of resistance.

Overall, these results underscore the urgent need for antimicrobial stewardship programs targeting livestock to curb the dissemination of resistant bacteria. Future research should focus on alternative strategies, such as probiotics and phage therapy, to reduce reliance on conventional antibiotics while ensuring animal health and food safety. In general, this chapter highlights the high prevalence of antibiotic-resistance genes in E. coli isolated from the intestines of sheep. The detection of multiple resistance genes, particularly those encoding sulfonamide and tetracycline resistance, suggests widespread antibiotic resistance. Additionally, the significant association between ESBL production and resistance to third- and fourth-generation cephalosporins underscores the need for stringent antibiotic stewardship in livestock management.

5. Conclusion

The presence of multiple antibiotic-resistance genes in intestinal bacteria isolated from sheep livestock represents a significant challenge to both animal and human health. These results highlight a growing issue of multidrug resistance among bacteria isolated from sheep intestines. The high prevalence of strains carrying multiple resistance genes underscores the urgent need for effective measures to reduce the spread of resistance and promote the rational use of antibiotics in agriculture and livestock farming. Alternative therapeutic strategies must be developed to preserve the effectiveness of antibiotics. There should be strict monitoring of antibiotic use in animal farming and the development of strategies to control the spread of resistant infections to ensure long-term food safety and human health. It is important to raise awareness and educate farmers, consumers, and health workers about the dangers of overuse of antibiotics and the importance of maintaining food safety through good hygiene practices.

Acknowledgments

The researchers would like to express their sincere thanks to the Custodian of the Two Holy Mosques Institute for Hajj and Umrah Research for supporting this project No (103/24).