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Introduction
Some strains of Escherichia coli (E. coli) possess pathogenic characteristics and can cause life-threatening diseases (Torres-León et al., 2022). The hemolytic capacity of E. coli is an important virulence factor, as the ability of the bacterium to lyse red blood cells is associated with the presence of oligomeric toxins capable of forming pores in the cell membrane. In livestock production, E. coli is responsible for significant economic losses (Torres-León et al., 2022). In humans, E. coli is associated with urinary and skin infections, meningitis, myositis, osteomyelitis, and orchiepididymitis (Vila et al., 2016).
Antibiotics such as aminoglycosides, penicillins, streptomycin, cephalosporins, sulfonamides, tetracyclines, and quinolones are commonly used to treat diseases caused by E. coli (Younis et al., 2017). However, many E. coli strains have developed resistance to these antibiotics (Roth et al., 2019). Approximately 20-45% of E. coli isolates exhibit resistance to first-line antibiotics such as tetracyclines, penicillins, and sulfonamides (Pitout, 2012). Although humans are the main carriers of antibiotic-resistant E. coli strains, antibiotic-resistant strains have also been reported in livestock species such as cattle, pigs, and poultry (Alonso et al., 2017). The presence of antibiotic-resistant microorganisms not only impacts animal health and welfare but also poses a serious public health concern, as over 60% of resistant bacteria originate from animals (Mcarthur, 2019).
Antibiotic resistance arises from a combination of natural and artificial factors, such as the high adaptability of microorganisms (Fernández-Rodríguez et al., 2020), their ability to exchange genetic material, the misuse of antibiotics (including self-medication and administration without veterinary supervision), incomplete treatments, and misdiagnoses (Torres-León et al., 2021). Common resistance mechanisms include antibiotic inactivation by enzymes, bacterial modifications, and alterations at the target site (Reygaert, 2018; Van Boeckel et al., 2015).
The remarkable adaptability of bacteria to extrinsic factors facilitates the development of multidrug resistance (Mendoza et al., 2019). Bacteria can acquire and transfer resistance genes through horizontal gene transfer mechanisms, including conjugation, transduction, and transformation. Genetic material is exchanged through plasmids, integrons, and transposons (Quiñones-Pérez, 2017). The transmission of resistance genes is a highly dynamic process driven by the movement of mobile genetic elements (Murray et al., 2022).
In intensive animal production systems, the widespread use of antimicrobials exerts selection pressure on microorganisms, leading to the accumulation and spread of resistance genes. E. coli can serve as a reservoir for resistance genes, making it a valuable sentinel organism for monitoring antimicrobial resistance trends in both animal and human populations (Loayza et al., 2020).
Antibiotic-resistant E. coli is recognized as one of the most pressing global public health concerns (Jang et al., 2017). The World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), and the World Organisation for Animal Health (OIE) have declared antimicrobial resistance a critical threat to humanity (WHO, 2014). Antibiotic resistance not only limits treatment options for infectious diseases but also increases the risk of pathogen transmission and outbreaks (Mcarthur, 2019; Quiñones-Pérez, 2017). While the incidence of E. coli-induced diarrhea has declined in developing countries, this disease continues to disproportionately affect children (Anderson et al., 2019). The objective of this study was to evaluate the antibiotic resistance of non-pathogenic E. coli strains isolated from pig farms.
Materials and Methods
Bacterial strains
Non-hemolytic Escherichia coli (γ-hemolytic) strains were obtained by rectal swabbing of 77 piglets randomly selected from six intensive swine farms in Valle del Cauca, Colombia. The selection criteria were based on farm records indicating the presence of diarrhea in piglets. Samples were collected in the cities of Yumbo (3° 34’ 56’’ N, 76° 29’ 29’’ W) and Palmira (3° 31’ 1’’ N, 76° 18’ 0’’ W). Piglets aged 4-40 days, weighing approximately 2-11.2 kg, were chosen, corresponding to the lactation or nursery phase. The methodology followed the protocols described in previous studies by Pabón-Rodríguez et al. (2023). The six bacterial isolates were designated as strains 1:2, 1:6, 2:1, 2:11, 3:2, and 3:12.
Hemolytic activity
The isolated strains were cultured in triplicate on blood agar plates supplemented with 5% defibrinated sheep red blood cells. The plates were incubated for 24 h at 37°C. Strains classified as γ-hemolytic did not exhibit red blood cell lysis around the colonies.
Molecular identification of strains
γ-Hemolytic E. coli strains were confirmed through PCR amplification of the 16S ribosomal RNA gene, using primers 27F and 1492R.
Gene amplification of LT, STa, and STb toxins
The presence of thermolabile (LT) and thermostable (STa and STb) toxin genes in E. coli strains was determined by PCR. To reactivate the strains, cryopreserved samples (-60°C) were thawed at room temperature (25°C). Briefly, 1 mL of thawed sample was inoculated into a test tube containing 4 mL of soy broth and incubated for 24 h at 37°C. Following incubation, the cultures were streaked onto MacConkey agar for isolation. E. coli strains that were γ-hemolytic and tested negative for the LT, STa, and STb genes were considered as non-pathogenic.
The presence of toxin genes was confirmed by visualizing the amplification bands of the target genes, with expected fragment sizes of STa: 163 bp, STb: 368 bp, and LT: 275 bp. PCR products were separated on 2% agarose gels via electrophoresis and visualized using photo-documentation equipment.
Antibiotic resistance of E. coli strains
The antimicrobial susceptibility of molecularly identified E. coli strains was assessed following the standardized disk diffusion method, measuring inhibition zone diameters (Begum et al., 2016).
Briefly, 50 µL of each bacterial inoculum was evenly spread on Mueller-Hinton agar using a sterile swab. Antibiotic discs were placed on the agar surface, including: ampicillin (10 µg), enrofloxacin (5 µg), ciprofloxacin (5 µg), gentamicin (10 µg), florfenicol (30 µg), fosfomycin (50 µg), doxycycline (30 µg), norfloxacin (10 µg), amikacin (30 µg), ceftiofur (30 µg), and apramycin (15 µg). The Petri dishes were then incubated at 37°C for 24 h.
At the end of the incubation period, standardized photographs were taken of the Petri dishes, and inhibition zones were analyzed using ImageJ software (Valencia-Hernández et al., 2016). Each antibiotic susceptibility test was performed in triplicate.
Results
Hemolytic Activity
The Escherichia coli strains did not exhibit a hemolytic halo around their colonies, indicating the absence of hemolysis. This is attributed to the lack of an enzyme system capable of lysing red blood cells (Sánchez-Neira and Angarita-Merchán, 2018). The hemolytic capacity of E. coli strains is associated with the expression of four genes in the hlyCABD operon (Soares et al., 2022). Hemolysis is a characteristic associated with pathogenicity (Sarowska et al., 2019). However, our results indicate that pathogenic E. coli strains can still carry virulence factors despite lacking hemolytic activity.
Molecular Identification of Strains
A fragment of the 16S rRNA gene of E. coli strains was amplified via PCR and subsequently sequenced. Figure 1 illustrates the amplification bands of the 16S rRNA gene from strains isolated via rectal swabs from piglets. Sequencing was performed using a reverse primer to generate two sequences per strain, minimizing the risk of false polymer formations.
The bands in lanes 1, 2, 3, 4, 5, and 6 appear at a higher position than the molecular marker lane (Mm), with a size of 1000 bp. Consequently, the observed band size was approximately 1500 bp, which is consistent with the expected size for the 16S rRNA gene in isolates 1:2, 1:6, 2:1, 2:11, 3:2, and 3:12, using molecular markers of 100 bp and 1 kb. Additionally, no contamination was observed in the reactions.
The use of growth promoters, antibiotics, and environmental conditions in animal husbandry influences the composition and abundance of microbial strains. These strains are commonly identified through variation in the 16S rRNA gene sequence (Mantilla and Torres, 2019).
Figure 1 PCR amplification of the 16S rRNA gene from E. coli strains isolated from piglets. Lanes (1-6) correspond to strains 1:2, 1:6, 2:1, 2:11, 3:2, and 3:12. Lane (Mm) contains the 100 bp molecular marker.
Table 1 presents the molecular identification results for strains 1:2, 1:6, 2:1, 2:11, 3:2, and 3:12, corresponding to partial 16S rRNA gene sequences. The percent identity with reference E. coli strains ranged between 94% and 98%, supporting the microbiological culture results.
Table 1 Genus and species identification of PCR fragments from E. coli strains isolated via rectal swabs from piglets.
| Encoded strain | E-value. | Identity percentage | Molecular identification | Length |
|---|---|---|---|---|
| 1:2 | 0,0 | 96,77% | E. coli Strain UT03 | 408pb |
| 1:6 | 0,0 | 97,78% | E. coli isolate RS16 | 406pb |
| 2:1 | 0,0 | 96,78% | E. coli Strain UT03 | 559pb |
| 2:11 | 0,0 | 98,02% | E. coli Strain wid10 | 605 pb |
| 3:2 | 0,0 | 94,04% | E. coli Strain LR09 | 688pb |
| 3:12 | 0,0 | 98,95% | E. coli isolate 68 | 286pb |
Amplification of LT, STa, and STb Genes
The E. coli strains (1:2, 1:6, 2:1, 2:11, 3:2, and 3:12) did not exhibit amplification of the LT (thermolabile), STa (thermostable type A), or STb (thermostable type B) toxin genes. Consequently, these strains are considered non-pathogenic.
Enterotoxigenic E. coli strains are a major cause of morbidity and mortality in neonatal pigs. Post-weaning diarrhea is associated with two primary virulence factors: adhesins (K88/F4, K99/F5, 987P/F6, F41/F7, and F18) and enterotoxins (LT, STa, and STb).
Antibiotic Resistance of E. coli Strains
Table 2 summarizes the antibiotic susceptibility profiles of E. coli strains (1:2, 1:6, 2:1, 2:11, 3:2, and 3:12), classified as resistant, intermediate, or susceptible.
The overall resistance profile of the strains was as follows: amikacin (20%), ceftiofur (20%), fosfomycin (20%), ciprofloxacin (40%), gentamicin (40%), florfenicol (80%), enrofloxacin (80%), norfloxacin (80%), apramycin (100%), ampicillin (100%), and doxycycline (100%).
Thus, the study strains exhibited multidrug resistance. Strain 1:2 demonstrated the highest resistance capacity, being resistant to 10 out of 11 tested antibiotics.
Table 2 Antibiotic resistance of E. coli strains 1:2, 1:6, 2:1, 2:11, 3:2, and 3:12
| Antimicrobial | Inhibition zone diameter (mm) | Reference (mm) | ||||
|---|---|---|---|---|---|---|
| 1:2 | 1:6 | 2:1 | 2:11 | 3:12 | ||
| Ampicillin | 0 | 0 | 0 | 0 | 0 | ≤ 13 |
| 14 - 16 | ||||||
| ≥ 17 | ||||||
| Ciprofloxacin | 11 | 22 | 28 | 23 | 8 | ≤ 19 |
| 20 - 21 | ||||||
| ≥ 22 | ||||||
| Gentamicin | 0 | 13 | 19 | 0 | 15 | ≤ 12 |
| 13 - 14 | ||||||
| ≥ 15 | ||||||
| Florfenicol | 0 | 0 | 0 | 16 | 0 | ≤ 14 |
| 15 - 18 | ||||||
| ≥ 19 | ||||||
| Fosfomycin | 0 | 26 | 32 | 21 | 25 | ≤ 12 |
| 13 - 15 | ||||||
| ≥ 16 | ||||||
| doxycycline | 0 | 3 | 3 | 4 | 7 | ≤ 12 |
| 13 - 15 | ||||||
| ≥ 16 | ||||||
| norfloxacin | 19 | 16 | 30 | 19 | 0 | ≤ 19 |
| 20 - 21 | ||||||
| ≥ 22 | ||||||
| Amikacin | 19 | 18 | 21 | 17 | 14 | ≤ 14 |
| 15 - 16 | ||||||
| ≥ 17 | ||||||
| Ceftiofur | 9 | 19 | 24 | 18 | 19 | ≤ 17 |
| 18 - 20 | ||||||
| ≥ 21 | ||||||
| Apramycin | 15 | 15 | 14 | 14 | 12 | ≤ 15 |
| 16-19 | ||||||
| ≥ 20 | ||||||
| Enrofloxacin Baytril | 15 | 10 | 24 | 14 | 0 | ≤ 16 |
| 17 - 22 | ||||||
| ≥ 23 | ||||||
Discussion
Resistance of Escherichia coli to fluoroquinolones (nalidixic acid, cinoxacin, pipemidic acid, enoxacin, ofloxacin, ciprofloxacin, pefloxacin, norfloxacin, lomefloxacin, levofloxacin, sparfloxacin, tosufloxacin, gatifloxacin, trovafloxacin, clinafloxacin, and moxifloxacin) is likely due to the acquisition and modification of plasmids that carry resistance genes (Singh et al., 2022). These results are consistent with those reported by Begum et al. (2016), who observed fluoroquinolone resistance in 84% of E. coli strains. This trend has also been reported in studies conducted in China and Japan (Terahara and Nishiura, 2019).
Previously, antibiotic-resistant E. coli strains isolated from pigs have been reported in Colombia (Mantilla et al., 2022). The observed resistance percentages were as follows: tetracycline (100%), sulfamethoxazole-trimethoprim (97.5%), ampicillin (95.2%), amoxicillin (83.1%), tylosin (82.1%), and florfenicol (74.6%) (Mantilla et al., 2022). These results align with the findings of the present study, particularly regarding resistance to ampicillin (100%) and florfenicol (80%). Additionally, the γ-hemolytic E. coli strains analyzed in this study were compared to a group of pathogenic β-hemolytic E. coli strains isolated from piglets, as reported by Pabón-Rodríguez et al. (2023). Both groups showed 100% resistance to ampicillin, apramycin, and doxycycline, as well as high resistance to enrofloxacin (80%) and florfenicol (80%).
The resistance of non-pathogenic E. coli strains to ampicillin and other antibiotics reflects the high prevalence of multidrug-resistant strains in the Colombian swine industry. This phenomenon is not unexpected, given the widespread availability and use of antibiotics in livestock production. The indiscriminate use of these drugs exerts selective pressure on bacterial populations, favoring the emergence and spread of resistant strains (Monger et al., 2021). A major contributing factor is the administration of subtherapeutic antibiotic doses in animal feed and drinking water. This practice creates an environment conducive to the development and dissemination of resistant bacteria (Garcias et al., 2024).
The high prevalence of multidrug resistance in Colombia underscores the urgent need to implement effective antimicrobial control and regulation strategies in the swine industry. This includes restricting the use of antibiotics for disease treatment and as growth promoters (Mantilla et al., 2022). Proper regulation is critical to minimizing the risk of disease outbreaks caused by resistant bacteria and safeguarding public health (Monger et al., 2021; Nowaczek et al., 2021; Daneman et al., 2023; Magiorakos et al., 2012).
Based on the results of this study, stricter monitoring and the adoption of responsible antibiotic management practices in pig farms are imperative to mitigate the risk of outbreaks and the spread of antibiotic-resistant bacteria. Therefore, regulatory enforcement by organizations such as the Colombian Agricultural Institute (ICA) is essential to ensure that antibiotics used in pig production are properly supervised and administered (Arenas and Melo, 2018).
The horizontal transfer of resistance genes among different bacterial populations highlights the need for continuous surveillance. The ability of E. coli to acquire and disseminate antimicrobial resistance genes makes it a key indicator for monitoring resistance trends in both animal and human populations (Loayza et al., 2020; O’Neill et al., 2023).
In Colombia, the ICA currently regulates the use of antibiotics in livestock production. However, these regulations must be strengthened and rigorously enforced to protect public health and ensure the long-term sustainability of the swine industry.
Conclusions
This study demonstrated that Escherichia coli strains isolated from pig farms exhibit resistance to multiple antibiotics. The strains were molecularly identified as non-hemolytic and lacked thermolabile (LT) and thermostable (STa and STb) toxins. The antibiotic resistance observed in these bacteria poses a risk to both animal and human health.
Therefore, it is crucial to implement stricter controls and regulations to mitigate the spread of antimicrobial resistance. The identification and characterization of these E. coli strains contribute to Colombia's national antimicrobial resistance response plan, as established by the Ministry of Health.
Our findings emphasize the need for stricter oversight in antibiotic use in swine production. The indiscriminate use of antibiotics should be reduced to prevent the selection and dissemination of resistant strains.
