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Article

Virulence and Antibiotic Resistance of aEPEC/STEC Escherichia coli Pathotypes with Serotype Links to Shigella boydii 16 Isolated from Irrigation Water

by
Yessica Enciso-Martínez
1,2,
Edwin Barrios-Villa
1,
Manuel G. Ballesteros-Monrreal
1,
Armando Navarro-Ocaña
3,
Dora Valencia
1,
Gustavo A. González-Aguilar
2,
Miguel A. Martínez-Téllez
2,
Julián Javier Palomares-Navarro
2 and
Fernando Ayala-Zavala
2,*
1
Departamento de Ciencias Químico-Biológicas y Agropecuarias, Universidad de Sonora, Caborca 83621, Mexico
2
Centro de Investigación en Alimentación y Desarrollo, A.C., Carretera Gustavo Enrique Astiazarán Rosas 46, Col. La Victoria, Hermosillo 83304, Mexico
3
Departamento de Salud Pública, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México 04510, Mexico
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(6), 549; https://doi.org/10.3390/pathogens14060549
Submission received: 9 April 2025 / Revised: 29 May 2025 / Accepted: 30 May 2025 / Published: 1 June 2025
(This article belongs to the Special Issue Virulence and Resistance Mechanisms in Multidrug-Resistant Gram-Negative Bacteria)
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Abstract

:
Irrigation water can serve as a reservoir and transmission route for pathogenic Escherichia coli, posing a threat to food safety and public health. This study builds upon a previous survey conducted in Hermosillo, Sonora (Mexico), where 445 samples were collected from a local Honeydew melon farm and associated packing facilities. Among the 32 E. coli strains recovered, two strains, A34 and A51, were isolated from irrigation water and selected for further molecular characterization by PCR, due to their high pathogenic potential. Both strains were identified as hybrid aEPEC/STEC pathotypes carrying bfpA and stx1 virulence genes. Adhesion assays in HeLa cells revealed aggregative and diffuse patterns, suggesting enhanced colonization capacity. Phylogenetic analysis classified A34 within group B2 as associated with extraintestinal pathogenicity and antimicrobial resistance, while A51 was unassigned to any known phylogroup. Serotyping revealed somatic antigens shared with Shigella boydii 16, suggesting possible horizontal gene transfer or antigenic convergence. Antibiotic susceptibility testing showed resistance to multiple β-lactam antibiotics, including cephalosporins, linked to the presence of blaCTX-M-151 and blaCTX-M-9. Although no plasmid-mediated quinolone resistance genes were detected, resistance may involve efflux pumps or mutations in gyrA and parC. These findings are consistent with previous reports of E. coli adaptability in agricultural environments, suggesting potential genetic adaptability. While our data support the presence of virulence and resistance markers, further studies would be required to demonstrate mechanisms such as horizontal gene transfer or adaptive evolution.

Graphical Abstract

1. Introduction

Water used for irrigation is a critical component of global agricultural systems, essential for crop development and food production. However, its microbiological quality has direct implications for food safety, public health, and environmental sustainability [1]. Using contaminated water in primary production is considered one of the major sources of fresh produce contamination [1]. Waste from dairy farms, including fecal matter, manure slurry, and wastewater, has been increasingly recognized as a significant environmental reservoir of antimicrobial-resistant and pathogenic E. coli. These residues often carry a high load of antibiotic-resistance genes and virulence-associated genes, which can disseminate through runoff or irrigation practices, contributing to the microbiological contamination of surface water sources used in agriculture [2,3]. The World Health Organization (WHO) and Food and Agriculture Organization (FAO) have reported that microbial contamination in fresh produce contributes to a significant proportion of foodborne illness outbreaks globally, with Escherichia coli being among the most frequent etiological agents [4]. In Latin America, approximately 70% of surface water used for irrigation in agricultural zones is untreated, increasing the likelihood of fecal contamination [5]. In this context, E. coli not only serves as a fecal indicator organism, but specific pathogenic variants, including diarrheagenic E. coli (DEC), pose a direct health risk due to their ability to cause gastroenteritis and severe systemic infections [6]. Among these, Enteropathogenic E. coli (EPEC) and Shiga-toxigenic E. coli (STEC) are especially relevant. EPEC strains are subclassified as typical (tEPEC), which carry the bfpA gene encoding the bundle-forming pilus, and atypical (aEPEC), which lack this gene [7]. On the other hand, STEC strains are responsible for severe diseases such as hemorrhagic colitis and hemolytic uremic syndrome. They are characterized by stx1 and/or stx2 genes, which encode Shiga toxins [8].
Moreover, the emergence of hetero-pathogenic or hybrid E. coli strains that combine virulence traits of different pathotypes (e.g., aEPEC/STEC) or of both DEC and ExPEC (extraintestinal pathogenic E. coli) represents a growing concern. These strains often display increased pathogenicity and resistance, complicating diagnosis and treatment [9]. In addition to pathotyping, serotyping remains a valuable tool in epidemiological surveillance. For example, WHO identified 12 serogroups classically associated with EPEC in 1987 [10], while serogroups such as O157, O26, and O111 are commonly linked to STEC. Recent reports have described E. coli strains sharing somatic antigens with Shigella boydii 16, including those isolated from pediatric patients with diarrhea and from animals raised for human consumption, suggesting horizontal gene transfer and a broader ecological niche [11].
Despite these advances, several knowledge gaps remain. Most surveillance studies focus on clinical or food isolates, with limited data on E. coli strains circulating in agricultural systems. Although relatively few studies have characterized environmental E. coli strains at both the genotypic and phenotypic levels, particularly regarding their hybrid virulence profiles and antimicrobial resistance, recent research has begun to address this gap. Despite these advances, several knowledge gaps remain. Most surveillance studies focus on clinical or food isolates, with limited data on E. coli strains circulating in agricultural systems. Although relatively few studies have characterized environmental E. coli strains at both the genotypic and phenotypic levels, particularly regarding hybrid virulence profiles and antimicrobial resistance [12], recent research has begun to address this gap. For instance, identified multidrug-resistant E. coli strains in the dairy farm environment carry diverse virulence-associated genes and antibiotic resistance determinants [2,13]. Similarly, whole-genome sequencing of ESBL-producing E. coli isolates from environmental sources revealed the co-occurrence of blaOXA−1, catB3, and arr-3 genes, along with transferable resistance elements and phylogenetic diversity [3]. This emerging body of evidence highlights the complexity of environmental E. coli and underscores the need for integrated surveillance approaches. This gap is significant given the role of irrigation water as a contamination route in fresh produce supply chains. This study builds upon previous work in Hermosillo, Sonora, Mexico [14], where 445 environmental samples were collected from a Honeydew melon farm and its packing facilities, leading to the isolation of 32 E. coli strains. Among these, strains A34 and A51, isolated from irrigation water, stood out due to their virulence and resistance profiles. These isolates were identified as aEPEC/STEC pathotypes and displayed serotype links to S. boydii 16, raising important questions regarding their origin, evolution, and potential public health risk. We hypothesize that the E. coli strains A34 and A51 may represent emerging hybrid pathotypes. The coexistence of virulence and resistance genes suggests the possible influence of environmental pressures and horizontal gene transfer, although the mechanisms were not directly investigated in this study. Based on their genetic makeup, adhesion phenotypes, and resistance to β-lactam antibiotics, these strains exemplify the evolutionary dynamics of E. coli in contaminated agricultural environments. The present study aims to address these gaps by providing a detailed molecular and phenotypic characterization of strains A34 and A51, thereby contributing to a better understanding of the microbial risks associated with irrigation water.

2. Materials and Methods

2.1. Bacterial Strains

This study builds upon a previous investigation conducted in Hermosillo, Sonora, Mexico, which focused on understanding the environmental reservoirs of pathogenic E. coli [14]. In that investigation, 445 environmental samples were collected from a Honeydew melon production system, including irrigation water, soil, packing materials, workers’ hands, and fruit surfaces. A total of 32 E. coli strains were isolated, of which 59% originated from irrigation water, highlighting its role as a primary contamination route in agricultural environments. Among these isolates, two strains, A34 and A51, were selected for detailed analysis due to their rare serological profile, specifically their somatic antigenic similarity with S. boydii 16, as determined by agglutination assays. This unusual finding suggests possible horizontal gene transfer or antigenic convergence between E. coli and Shigella species, a phenomenon that is infrequently reported in environmental isolates. Because of their unique serotype identity, these strains were particularly interesting for further characterization.

2.2. Isolation and Biochemical Characterization of E. coli

The samples were inoculated on MacConkey and Eosin Methylene Blue (Becton, Dickson and Company, Sparks, Baltimore, MD, USA) and incubated for 18 h at 37 ± 2 °C. Following that, biochemical characterization was confirmed through a series of standard biochemical assays, which included mobility, indole, sulfhydric acid production, glucose fermentation, lactose fermentation, Simmons citrate, Voges-Proskauer, urea, gas production, methyl red, and lysine decarboxylase [15].

2.3. Genomic DNA Extraction

The E. coli strains displaying the biochemical characteristics were processed for DNA extraction using the alkaline lysis method following the established protocols in The Molecular Cloning Laboratory Manual 2012 [16]. The extracted DNA was preserved at −20 °C for preservation.

2.4. Molecular Identification of E. coli

The E. coli strains were identified using a conventional PCR with GoTaq Green Master Mix (Promega, Madison, WI, USA) targeting the ybbW (Table S1), which encodes the allantoin transporter protein and is exclusive to this species [17,18]. The amplified PCR product was visualized via electrophoresis on a 1% agarose gel in 1× TAE buffer with GelStarTM Stain (Lonza, Morristown, NJ, USA). After confirmation of the identification, the E. coli strains were maintained in Luria-Bertani broth (Becton, Dickson and Company, Sparks, MD, USA) containing 20% v/v glycerol and stored at −80 °C [19].

2.5. Determination of Diarrheagenic Pathotypes

Conventional PCR was performed to search for genetic pathotype markers: the eaeA gene for EPEC/EHEC, the bfpA gene for EPEC, the stx1 and stx2 genes for EHEC/STEC, thermolabile (LT) and thermostable (ST) toxins for ETEC, the daaE and afa/draBC genes for DAEC, the ial for EIEC, and the pCVD for EAEC identification using specific primers (Table S1).

2.6. Adherence Assay

HeLa cells were cultured in six-well polystyrene plates containing sterile glass coverslips, using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), and incubated at 37 °C in a 5% CO2 atmosphere until reaching approximately 80% confluence. A cell suspension of 5 × 10⁴ cells/mL was then prepared in 2 mL of antibiotic-free DMEM supplemented with 10% FBS and incubated overnight under the same conditions.
After incubation, HeLa monolayers were gently washed with sterile phosphate-buffered saline (PBS), and 2 mL of fresh DMEM with 10% FBS was added to each well. A 0.5 McFarland suspension of each E. coli isolate, prepared from an 18–24 h preculture in brain heart infusion broth, was diluted in DMEM and applied to the HeLa cells at a multiplicity of infection (MOI) of 30:1 (bacteria: cell). The co-cultures were incubated for 3 h at 37 °C with 5% CO2.
Following incubation, the monolayers were washed with PBS to remove non-adherent bacteria, fixed with methanol, stained with Giemsa, and washed three times with PBS. Coverslips were removed and mounted on microscope slides for observation under light microscopy at 1000× magnification. E. coli strain K-12 (commensal) was a negative adherence control. Each isolate was evaluated in three independent assays, and adherence patterns were classified qualitatively as aggregative, diffuse, or localized, based on previously described morphological criteria [19].

2.7. Serotyping

To establish a serotype-based classification of the strains, agglutination assays were conducted utilizing 96-well microtitre plates, and rabbit serum (SERUNAM) was obtained a2.1 Bacterial strains against 186 somatic antigens and 53 flagellar antigens for E. coli, as well as 45 somatic antigens for Shigella species, following the methods described by Ørskov and Ørskov [20].

2.8. Phylogenetic Group Determination

The phylogenetic group of the identified strains was determined using the scheme previously proposed by Clermont in 2013 [21] (Table S1).

2.9. Virulence-Associated Genes

Eleven virulence genes associated with 9 virulence factors were evaluated: cytotoxic necrotizing factor (cnf-1), secreted autotransporter toxin (sat), type II capsule synthesis (kpsMII), catecholate siderophore receptor (iroN), adhesins (afa and afa/draBC), type P pilus (papGII and papGIII), cerebral endothelial invasion (ibeA), hemolysin A (hlyA) and total distension toxin (cdtB). Conventional PCR was used using GoTaq Green MasterMix (Promega) and specific primers for each gene (Table S1). The PCR products were resolved on a 1% agarose gel [19]. CFT073 and J96 were used as positive controls.

2.10. Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR)

To establish a phylogenetic relationship, an ERIC-PCR was performed using previously reported primers (Table S1) [22]. The reaction tube contained 0.4 μM of each primer, GoTaq MasterMix (Promega Corporation, Madison WI, USA) (containing reaction buffer, 400 μM of each dNTP, 3 mM MgCl2, and 1× Taq® DNA polymerase) to a final volume of 12 μL in a MiniAmp Plus thermal cycler (Applied BioSystems, Foster City, CA, USA). The protocol includes a denaturation cycle at 95 °C followed by 35 denaturation cycles at 95 °C for 60 s, 50 °C for 60 s, and 72 °C for eight minutes, and a final extension cycle for 16 min. The amplicons were resolved in 1% agarose gel electrophoresis and ethidium bromide to visualize them using a UVP transilluminator (AnalytikJena, Beverly, MA, USA). The distance matrices were first obtained with Phyelph 1.4 by placing the bands in each gel lane. A 5% distance between bands was considered part of the same cluster for the clustering, and the UPGMA algorithm and the Dice coefficient were used. A csv file of the distance matrices was then generated with PhyloM to make the dendrogram in MEGA 11.

2.11. Antibiotic Susceptibility

The Kirby-Bauer disc diffusion method was used to conduct the antibiogram, in which the commonly prescribed antibiotics for E. coli infections were tested. These included amikacin (30 µg), ampicillin (10 µg), aztreonam (30 µg), cefotaxime (31 µg), cefuroxime (30 µg), ceftriaxone (32 µg), cefepime (30 µg), ciprofloxacin (5 µg), ertapenem (10 µg), amoxicillin/clavulanic acid (20/10 µg), meropenem (10 µg), and sulfamethoxazole/trimethoprim (1.25/23.75 µg). The E. coli strains of our study and E. coli ATCC 25922 (control) were cultured in 5 mL Mueller Hinton broth and incubated at 35 °C ± 2 °C for 16–18 h. The diameters of the inhibition zones were measured in millimeters, and interpretation of susceptibility (susceptible, intermediate, or resistant) was performed according to the Clinical and Laboratory Standard Institute (CLSI) [23], using the standardized zone diameter breakpoints provided for each antimicrobial tested. Furthermore, Magiorakos’ criteria were followed to categorize the E. coli strains as non-multidrug-resistant (NMDR), multidrug-resistant (MDR), and extremely drug-resistant (XDR).

2.12. Molecular Identification of Extended Spectrum β-Lactamases (ESBLs) and Non-ESBL Genes

The E. coli strains were examined for the presence of antibiotic-resistance genes using conventional PCR. Specific primers for ESBL genes (blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaCTX-M-151, blaTEM, blaSHV) and non-ESBL genes (qepA and aac(6′)-lb-cr) were used and listed in Table S1. Each PCR reaction contained 12.5 µL GoTaq Green Master Mix (Promega Corporation, Madison, WI, USA), 0.5 µL of each primer (10 µM), 1.5 µL of template DNA (50–75 ng), and nuclease-free water to make a final volume of 25 µL. The PCR product was then examined through electrophoresis on a 1% agarose gel.

3. Results and Discussion

3.1. Diarrheagenic Pathotypes

A previous study conducted in Hermosillo, Sonora [14] examined 445 samples obtained from a local farm and packing facilities of Honeydew melon. The results revealed the presence of 32 E. coli strains. Among these, two specific strains, A34 and A51, were identified, isolated from irrigation water, and characterized by their high pathogenicity. These strains contained the bfpA and stx1 genes, suggesting they belong to the aEPEC/STEC pathotype (Table 1). To better understand the adherence behavior of the isolates, assays were performed on HeLa cell monolayers. Strains A34 and A51 exhibited distinct adherence patterns, classified qualitatively as aggregative and diffuse (Figure 1). Specifically, Figure 1a shows a compact and dense clustering of bacterial cells on the surface of HeLa, indicative of a strong aggregative adherence pattern. In contrast, Figure 1b illustrates a more scattered distribution of bacteria across the cell monolayer, consistent with a diffuse adherence pattern, although bacterial attachment remains evident. The presence of bfpA and stx1 genes suggests that these strains may possess virulence potential associated with aEPEC and STEC pathotypes. However, functional expression of these genes was not assessed, and the actual impact on pathogenicity remains to be determined. The observed aggregative and diffuse adherence patterns in HeLa cells are consistent with previously described phenotypes, but further studies using host-relevant models (e.g., intestinal organoids or animal infections) are necessary to confirm their role in disease.
The hetero-pathogens aEPEC/STEC of the E. coli strains A34 and A51 show combinations of virulence factors. These hetero-pathogens are strictly enteropathogenic, identified by diarrheagenic E. coli pathotypes associated with specific virulence factors [8]. Detecting hetero-pathogenic E. coli strains in various contexts, including the environment and animals but predominantly in human infections, highlights their relevance. For example, a study in Limpopo, South Africa, showed that 25.3% of children hospitalized for diarrhea had the EAEC/ETEC pathotype [24]. Similarly, in Sweden, the STEC/ETEC pathotype incidence in a clinical collection was 2.05% [25]. Therefore, the identification of EAEC/ETEC and STEC/ETEC pathotypes highlights the dynamic evolution of virulence in E. coli, raising concerns about their potential to cause more severe or persistent infections. However, the actual impact of combined pathogenic mechanisms in these strains requires further investigation.
Environmental water is a significant vector for these strains. In Johannesburg, South Africa, several hetero-pathogenic strains were identified, including EHEC/ETEC (1.8%), EAEC/aEPEC (7.6%), EPEC/ETEC (2.4%), EAEC/ETEC (3%), and EPEC/EHEC (1.8%) [26]. In the animal context, hetero-pathogenic pathotypes have been documented in pigs, as indicated by a study in Spain, where 8.1% of ETEC/STEC was identified [27]. Additionally, in Mexico, 4.5% of STEC/ETEC was observed in wild animals (deer), suggesting that these animals can be reservoirs of pathogenic E. coli strains [28], raising the possibility of transfer of these strains to the environment, including irrigation water. Runoff from feces, combined with inadequate water management practices, increases the probability of pathogen transfer to crops, potentially leading to foodborne illnesses [29]. This highlights the potential for these pathotypes to spread beyond animal reservoirs, potentially contaminating environmental water sources and posing a significant risk to both public health and agricultural practices.
Various microbiological quality control strategies have been developed and implemented to mitigate the risk of contamination by heteropathogenic E. coli strains in irrigation water. These strategies include membrane filtration, chlorine dioxide treatment, ultraviolet radiation, ozone disinfection, and sodium hypochlorite chlorination [30]. Applying these preventive measures is crucial to ensure the safety of agricultural products intended for human consumption. Good Agricultural Practices (GAP) are also essential in preventing pathogenic contamination in food production. GAPs related to irrigation water management, such as risk assessments, implementing appropriate irrigation systems, and periodic microbiological testing of agricultural water, are vital to reducing the risk of pathogen transmission through crops [31]. Using E. coli to indicate fecal contamination and the potential presence of human enteric pathogens in irrigation water effectively manages food safety [32]. Since E. coli can persist in the environment for extended periods, its detection and monitoring provide valuable insights into the microbiological quality of water used in agriculture, helping prevent foodborne illnesses. Over the years, these strategies have proven effective to some extent; however, challenges remain in terms of sensitivity, cost, and speed, particularly in large-scale agricultural settings. Recent advancements, such as rapid on-site diagnostic tools and real-time monitoring systems, hold promise for improving detection accuracy and reducing delays in identifying contamination. In the future, the integration of these innovative technologies with environmental data analysis could lead to more effective and proactive strategies for managing water quality, ultimately reducing the risk of foodborne diseases and ensuring safer agricultural practices.

3.2. Serotyping

During the serotyping of E. coli strains from irrigation water, it was found that strain A34 belonged to serogroup H7: O **, while strain A51 belonged to H-:O ** (Table 1). Both strains shared an identity with the somatic antigen of S. boydii 16, as demonstrated by serological tests (Table 2). However, unlike S. boydii 16, which is immobile due to a deletion in the flhDC flagellar operon [33], E. coli strains A34 and A51 were mobile. Only strain A34 exhibited the specific flagellar antigen (H7), detected through serological assays. The H7 flagellar antigen plays a crucial role in the colonization and pathogenicity of the strains in the host. This antigen, related to flagellin, triggers an immune response by stimulating the secretion of pro-inflammatory chemokines in human intestinal cells. The expression of flagellar genes is regulated in response to specific environmental signals, inhibiting flagellar biosynthesis to conserve energy and minimize detection by the host immune system [34].
Furthermore, it was discovered that the somatic antigen of E. coli strains A34 and A51 was identical to that of S. boydii 16. This phenomenon can be explained by the presence of common or closely related somatic antigens between E. coli and Shigella serotypes, which has been documented. For example, strains of S. boydii 1, 2, 4, 5, 8, 11, 14, and 15 cross-react with strains of E. coli O149, O87, O53, O79, O143, O105, O32, and O112, respectively [35]. In a study conducted by Liu et al., the presence of the wzx (flippase) and wzy (polymerase) genes in E. coli was identified, confirming the existence of a shared epitope similar to that found in the O antigen of the S. boydii 16 pentasaccharide unit. The presence of S. boydii 16-like somatic antigens in E. coli may reflect genetic similarities and possibly shared epitopes. While such antigenic convergence could influence bacterial properties, our study does not directly assess their biological or pathogenic consequences. This modification could significantly complicate the identification and classification of infections caused by these strains, necessitating the development of more specific diagnostic tools and approaches to detect and differentiate these infections from others caused by traditional E. coli pathotypes.

3.3. Phylogenetic Group Determination

Regarding identifying the phylogenetic group to which the E. coli strains isolated from irrigation water belonged, it was found that the A34 strain presented a B2 phylogenetic group; however, A51 did not achieve any group identification (Table 1). The B2 phylogenetic group of E. coli has diverse pathogenic potential and contains commensal but also includes intestinal and extraintestinal pathogenic isolates. The genetic profile of the B2 phylogenetic group of E. coli has been associated in the literature with horizontal gene transfer and accumulation of virulence traits. Although we observed virulence and resistance markers in our isolates, our study did not include genomic analyses to confirm such events [36]. The strains belonging to the B2 phylogenetic group of E. coli have been observed to have more virulence genes and genes related to antibiotic resistance than other phylogenetic groups of E. coli. It is thought that the acquisition of these genes could have resulted from horizontal gene transfer events. Assigning E. coli strains to a particular phylogroup provides insight into their ecological niche, lifestyle, and propensity to cause disease [37]. Understanding the phylogenetic distribution of E. coli strains, particularly the higher prevalence of virulence and antibiotic resistance genes in the B2 group, is essential for advancing both diagnostic methods and broader public health strategies. This knowledge enables the development of more precise tools for early detection, tailored interventions, and effective monitoring programs across healthcare, agriculture, and environmental settings.
The prevalence of E. coli phylogenetic group B2 in agricultural samples found in primary production environments, such as irrigation water, agricultural soils, and fresh produce, has been reported in previous studies. Corzo et al. [38] reported the presence of E. coli belonging to the phylogenetic group B2 in Northern Mexico in irrigation water used for tomato crops (2%) and in jalapeño pepper crop soil (11.1%). This is also consistent with the findings of Jonhson et al. [39], who reported that only 5% of E. coli strains isolated from surface waters from Minnesota and Wisconsin belonged to phylogroup B2. Wild animals can be carriers of E. coli strains, such as those characterized in a deer population in Mexico, in which it was found that 13.6% belonged to the phylogenetic group B2 [28]. Similarly, in China, E. coli B2 has been detected in 26.7% of farmed ducks from farms in the Zhanjiang area, mainly carrying the blaTEM gene [40].
Phylogenetic analysis of E. coli strains isolated from irrigation water revealed the presence of group B2, known for its pathogenic potential and antibiotic resistance. These results underscore the importance of monitoring irrigation water quality and understanding human-animal-environment interactions in the spread and genetic diversity of E. coli, contributing to a better understanding of public health and food safety risks.

3.4. Virulence-Associated Genes and ERIC-PCR

Another essential feature is that the A51 strain of E. coli presented the afa/draBC genetic encoding for the Afa/DraBC adhesin (Table 3). Several virulence factors have been identified in the accessory genome of E. coli, encoding proteins involved in adhesion, invasion, motility, delivery of effector molecules, and toxicity. In the A51 strain of E. coli, the presence gene afa/draBC encodes the adhesin Afa/DraBC, which mainly allows the bacterium to attach to host cells. Specifically, Afa/DraBC makes it easier for E. coli to bind to cells in the urinary and gastrointestinal tract, allowing it to colonize and establish an infection in the host. In addition to its adhesive function, Afa/DraBC has been shown to play a role in phagocytosis resistance and survival in harsh environments. Not all strains of E. coli contain Afa/DraBC, but those that can be pathogenic and cause infections in humans and animals contain them [41]. This fimbrial adhesin also contributes to biofilm formation, a key factor in chronic and recurrent infections, particularly in urinary tract infections and gastrointestinal diseases. These mechanisms by which Afa/DraBC enhances bacterial survival and virulence could lead to the development of targeted therapeutic strategies or vaccines aimed at disrupting its function, offering the potential for more effective treatments against E. coli-induced infections.
The afa/draBC gene has been reported to be mobilizable via horizontal gene transfer in other E. coli strains [42]. While its presence in A51 suggests the possibility of such genetic events, our study does not provide direct evidence of the transfer mechanism involved. In addition, some studies suggest that afa genes can be transferred between different strains of E. coli via mobile DNA elements, such as plasmids or transposons [43].
Several studies have demonstrated the presence of the afa/draBC gene in E. coli strains. Such is the case reported by Mihailovskaya et al. [44], who reported the presence of this gene in 61.2% of isolates from healthy cows and calves in Perm Krai. The presence of the afa/dra gene has also been reported in chickens (1.25%) and swine (2.29%) farmed in South Korea [45]. Since wild animals can carry these strains and have been found in agricultural soil samples and fresh produce, it is plausible that they can also be transported into irrigation water through various pathways, such as fecal deposition of contaminated animals, runoff of contaminated water from agricultural areas, or direct deposition of bacteria in water. Therefore, E. coli strains containing the afa/draBC gene in irrigation water could pose a potential risk to public health if these strains survive and persist in that environment, mainly if used to irrigate crops intended for human consumption.
In addition, to establish a phylogenetic relationship between E. coli strains A34 and A51, a phylogeny based on ERIC-PCR was performed (Figure 2). It was observed that the A32 and A51 strains presented a similarity of more than 80% according to their ERIC profiles. The A34 was also quite similar, but there was more variation in the ERIC profiles, so it is in another branch. According to the ERIC profile, E. coli ATCC 25922 is far removed from its isolates; this is interesting since 25922 is considered uropathogenic according to its virulence gene load. These results suggest genetic differences between the studied strains, which could affect their pathogenicity and virulence capacity.

3.5. Antibiotic Susceptibility and Extended-Spectrum Β-Lactamases Genes

E. coli strains A34 and A51 resisted various antibiotics, including cephalosporins, carbapenems, beta-lactams, quinolones, penicillins, and sulfonamides (Table 4). These strains carried blaCTX-M-151 (A34) and blaCTX-M-9 (A51), which are associated with extended-spectrum β-lactamase production and resistance to cephalosporins and other β-lactam drugs. Although these genes were detected by conventional PCR, no quantitative gene expression analysis or functional assays were performed. Therefore, while their presence suggests a potential role in resistance, further studies are needed to confirm their expression and enzymatic activity in these isolates. It is well known that E. coli develops antibiotic resistance through various mechanisms, including acquiring genes encoding specific enzymes [46]. This study observed that E. coli strains A34 and A51 did not have genes related to quinolone resistance. Therefore, this resistance could be due to other mechanisms, such as upregulation of efflux pumps or mutations in DNA gyrase or topoisomerase IV [47]. Among the significant mutations are those in the gyrA, gyrB, parC, and parE genes [48]. However, the most mutated residues in ciprofloxacin-resistant strains are serine and aspartic/glutamic acid in the gyrA/parC IV helix [49]. The identification of these mutations could provide important insights into the molecular basis of quinolone resistance, which could facilitate the development of novel diagnostic tools or therapies targeting these resistance mechanisms. Additionally, understanding how these mutations evolve and spread across bacterial populations is crucial for managing antibiotic resistance and preventing the emergence of multidrug-resistant strains.
The selection of antibiotics used in food production is geographically variable and is influenced by factors such as the production system, the type of agriculture, and the legislation in force. The indiscriminate use of antibiotics in agriculture and the release of these compounds into the environment through various sources, such as human waste and their application in agriculture, has resulted in a prolonged presence of antibiotics in the environment. This prolonged exposure has led to the emergence of resistant bacteria [50]. Bacteria that were initially susceptible to antibiotics have developed resistance, primarily through modification of their target binding sites, enzyme neutralization, or membrane permeability changes induced by efflux pumps. In addition, bacteria can acquire antibiotic-resistance genes from other bacteria or phages through horizontal gene transfer [51].
The use of antibiotics in agriculture raises concerns because of the risk of spreading resistance to clinically significant bacteria. Accordingly, the Food and Agriculture Organization of the United Nations has recommended improved awareness, capacity building, monitoring, and management of antimicrobial use in food and agriculture, as well as the promotion of good practices in food and agricultural systems and the prudent use of antimicrobials in its plan to combat antimicrobial resistance [52]. The environment serves as a reservoir of antibiotic resistance genes transferable to clinically significant pathogenic bacteria, providing a diversified gene pool [53].
Irrigation water represents a significant source of contamination of fresh produce with antibiotic-resistant bacteria. This phenomenon is rising due to the selective pressure exerted by anthropogenic factors [54]. Aquatic ecosystems have been identified as primary reservoirs of antibiotic-resistant bacteria, and determinants of resistance in these environments have been documented.
A study by Bolukaoto et al. [26] reported the presence of E. coli strains resistant to cefuroxime (100%), ceftazidime (86%), and cefotaxime (81%) in ambient water in Johannesburg, South Africa. Likewise, Montero et al. [55] identified the presence of extended-spectrum beta-lactamase (ESBL) producing E. coli in 58% of isolates obtained from irrigation water used to produce horticultural products in Ecuador. Similarly, Gekenidis et al. [54] demonstrated the presence of 22% E. coli ESBL in isolates collected from irrigation water in various horticultural areas in Switzerland. Agricultural irrigation water in Valencia, Spain, showed a high prevalence of multidrug-resistant E. coli strains (70.4%), and strains had the blaTEM gene (96%). Likewise, these strains showed resistance to sulfonamides (93.3%), quinolones (73.3%), and tetracyclines (66.7%) [56]. This indicates that regular monitoring of irrigation water is required, including parameters related to antibiotic resistance. Similarly, multi-resistant strains of E. coli (17%) were detected in irrigation water of food products in Northwestern Mexico, which showed excellent resistance to cefotaxime (48.3%), ampicillin (44.8%), and tetracyclines (37.9%) [57]. This represents a potential source of human infection, so routine monitoring of irrigation water from food crops should be performed.
Regarding resistance isolated from farm animals, Mihailovskaya et al. [44] found that 32.7% of E. coli strains isolated from healthy cattle were shown to be multidrug-resistant to at least three groups of antibiotics. In addition, beta-lactam resistance genes were identified in significant proportions: blaTEM (100%), blaSHV (31.6%), and blaCTX-M (26.3%). These findings underscore the urgency of addressing and monitoring antimicrobial resistance, as it poses risks to human and animal health, highlighting the importance of genetic characterization for effectively managing this problem. Several anthropogenic activities have been reported as sources of irrigation water pollution, including animal intrusions and the discharge of poultry and pig effluents [58].
The identification of hybrid E. coli strains harboring virulence and resistance genes in irrigation water supports the relevance of the One Health approach. Irrigation water represents a convergence point for environmental, agricultural, and human activity, facilitating the potential transmission of pathogenic and antimicrobial-resistant bacteria across ecosystems. These findings underscore the need for integrated monitoring systems that include environmental samples alongside clinical and veterinary surveillance. Addressing such microbial threats requires coordinated action across sectors to mitigate risks to public health, food safety, and ecological stability.
This study presents several limitations that should be acknowledged. First, the molecular characterization of the strains was limited to conventional PCR, without functional validation such as gene expression analysis or phenotypic assays for toxin production or antibiotic degradation. Second, the classification of hybrid pathotypes and inference of horizontal gene transfer were based on gene detection and phenotypic traits, without genomic confirmation through whole-genome sequencing, which would be necessary to assess gene synteny, mobility elements, and epitope-level comparisons, particularly in relation to the observed antigenic similarity with Shigella boydii 16. In addition, the use of ERIC-PCR provided only a low-resolution overview of phylogenetic relationships among the isolates. Lastly, the adhesion assay on HeLa cells was qualitatively described, lacking quantitative scoring or molecular insight into host-pathogen interaction mechanisms.
Future studies should address these limitations by incorporating whole-genome and transcriptomic approaches to validate the genetic context of virulence and resistance markers. Functional assays such as ELISA, reporter systems, or in vivo infection models (e.g., intestinal organoids or murine models) are also recommended to evaluate the pathogenic potential of the isolates. Moreover, expanding the number of strains studied from diverse agricultural sources would provide a more comprehensive understanding of environmental E. coli diversity and risk. These efforts will strengthen the evidence base for integrated microbial risk assessments in farming systems and align with the One Health approach to food safety and public health.

4. Conclusions

This study highlights the presence and characterization of heteropathogenic strains of E. coli (aEPEC/STEC) isolated from irrigation water. The A34 and A51 strains showed aggregative and diffuse adhesion patterns, with the A34 strain belonging to the phylogenetic group B2, known for its pathogenic potential and antibiotic resistance. Both strains shared somatic antigens with S. boydii 16, suggesting possible horizontal gene transfer events. In addition, these strains were resistant to multiple antibiotics, including cephalosporins and β-lactams, due to the presence of the blaCTX-M-151 and blaCTX-M-9 genes. Identifying antibiotic-resistant E. coli strains in irrigation water is a public health concern, especially in the context of contamination of fresh produce. The finding underscores the importance of implementing systematic water quality monitoring programs, particularly microbiological indicators and resistance profiles. Strengthening surveillance policies can help detect emerging strains early and reduce the risk of transmission through the food chain. Moreover, integrating Good Agricultural Practices, regular water treatment protocols, and education on the responsible use of antibiotics is critical to mitigating the dissemination of resistant bacteria. Ensuring the microbiological safety of water used in food production is essential to protect consumer health and maintain the integrity of the food supply system

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens14060549/s1, Table S1: Specific oligonucleotides were used in this study. References [10,17,18,22,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74] are cited in the supplementary materials.

Author Contributions

Conceptualization, Y.E.-M., M.A.M.-T. and F.A.-Z.; methodology, Y.E.-M., E.B.-V., M.G.B.-M. and A.N.-O.; formal analysis, Y.E.-M., E.B.-V., M.G.B.-M., A.N.-O. and F.A.-Z.; investigation, Y.E.-M., E.B.-V., M.G.B.-M., D.V., J.J.P.-N. and G.A.G.-A.; resources, F.A.-Z. and E.B.-V.; data curation, Y.E.-M., E.B.-V. and D.V; writing—original draft preparation, Y.E.-M., E.B.-V., M.G.B.-M., J.J.P.-N. and F.A.-Z.; writing—review and editing, Y.E.-M., E.B.-V., M.G.B.-M., J.J.P.-N. and F.A.-Z.; supervision, M.A.M.-T., G.A.G.-A. and F.A.-Z.; project administration, F.A.-Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The dataset could be available on request.

Acknowledgments

The Consejo Nacional de Humanidades, Ciencias y Tecnología, is thanked for the scholarship to Enciso-Martínez for doctoral studies.

Conflicts of Interest

The authors declare no conflict of interest.

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Pathogens 14 00549 g001
Figure 1. Pattern of aggregative and diffuse adhesion in E. coli strain A34 (a) and A51 (b).
Figure 1. Pattern of aggregative and diffuse adhesion in E. coli strain A34 (a) and A51 (b).
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Figure 2. Dendogram of E. coli strains (A32, A34, A51, and ATCC 25922) was obtained by clustering ERIC profiles using the UPGMA algorithm and the DICE similarity coefficient. PG: Phylogenetic group; RG: Resistance genotype. ** indicates a somatic antigen (O) related to Shigella boydii 16.
Figure 2. Dendogram of E. coli strains (A32, A34, A51, and ATCC 25922) was obtained by clustering ERIC profiles using the UPGMA algorithm and the DICE similarity coefficient. PG: Phylogenetic group; RG: Resistance genotype. ** indicates a somatic antigen (O) related to Shigella boydii 16.
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Table 1. Pathotypes, serotyping, and phylogenetic groups of E. coli strains.
Table 1. Pathotypes, serotyping, and phylogenetic groups of E. coli strains.
StrainSourcePathotypeSerotypingPhylogenetic Group
A34Irrigation wateraEPEC/STECH7: O**B2
A51Irrigation wateraEPEC/STECH-: O**Unknown
**: Somatic antigenic relation with Shigella boydii 16.
Table 2. Agglutination titers of unabsorbed and absorbed E. coli A34, A51, 64474, 0179, 0188, and S. boydii 16 sera.
Table 2. Agglutination titers of unabsorbed and absorbed E. coli A34, A51, 64474, 0179, 0188, and S. boydii 16 sera.
AntigenTiters of Unabsorbed SeraTiters of Sera Absorbed with Boiled Cultures
E. coli 64474 absorbed with:S. boydii 16 absorbed with:
E. coli 64474E. coli 0179E. coli 0188S. boydii 16E. coli 0179E. coli 188S. boydii 16E. coli 64474E. coli 0179E. coli 0188
E. coli A341:100--1:800----1:8001:1600
E. coli A511:100--1:800----1:8001:1600
Table 3. Virulence-associated genes of the E. coli A34 and A51.
Table 3. Virulence-associated genes of the E. coli A34 and A51.
Straincnf-1satPkpsMIIafa operoniroNafa/draBCpapGIIpapGIIIibeAhlyAcdtB
A34-----------
A51-----+-----
Table 4. Antibiotic susceptibility and extended-spectrum β-lactamases genes of the E. coli A34 and A51.
Table 4. Antibiotic susceptibility and extended-spectrum β-lactamases genes of the E. coli A34 and A51.
StrainResistotypeESBL GenesESBL Non-Genes
A34CTX, CXM, CRO, FEP, MEM, ETP, AMC, AMP, CIP, STXblaCTX-M-151--
A51CTX, CXM, CRO, FEP, MEM, ETP, AMC, AMP, CIP, SXTblaCTX-M-9--
Cefotaxime (CTX); cefuroxime (CXM); ceftriaxone (CRO); cefepime (FEP); meropenem (MEM); ertapenem (ETP); amoxicillin/clavulanic acid (AMC); ampicillin (AMP); ciprofloxacin (CIP); and sulfamethoxazole/trimethoprim (STX).
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Enciso-Martínez, Y.; Barrios-Villa, E.; Ballesteros-Monrreal, M.G.; Navarro-Ocaña, A.; Valencia, D.; González-Aguilar, G.A.; Martínez-Téllez, M.A.; Palomares-Navarro, J.J.; Ayala-Zavala, F. Virulence and Antibiotic Resistance of aEPEC/STEC Escherichia coli Pathotypes with Serotype Links to Shigella boydii 16 Isolated from Irrigation Water. Pathogens 2025, 14, 549. https://doi.org/10.3390/pathogens14060549

AMA Style

Enciso-Martínez Y, Barrios-Villa E, Ballesteros-Monrreal MG, Navarro-Ocaña A, Valencia D, González-Aguilar GA, Martínez-Téllez MA, Palomares-Navarro JJ, Ayala-Zavala F. Virulence and Antibiotic Resistance of aEPEC/STEC Escherichia coli Pathotypes with Serotype Links to Shigella boydii 16 Isolated from Irrigation Water. Pathogens. 2025; 14(6):549. https://doi.org/10.3390/pathogens14060549

Chicago/Turabian Style

Enciso-Martínez, Yessica, Edwin Barrios-Villa, Manuel G. Ballesteros-Monrreal, Armando Navarro-Ocaña, Dora Valencia, Gustavo A. González-Aguilar, Miguel A. Martínez-Téllez, Julián Javier Palomares-Navarro, and Fernando Ayala-Zavala. 2025. "Virulence and Antibiotic Resistance of aEPEC/STEC Escherichia coli Pathotypes with Serotype Links to Shigella boydii 16 Isolated from Irrigation Water" Pathogens 14, no. 6: 549. https://doi.org/10.3390/pathogens14060549

APA Style

Enciso-Martínez, Y., Barrios-Villa, E., Ballesteros-Monrreal, M. G., Navarro-Ocaña, A., Valencia, D., González-Aguilar, G. A., Martínez-Téllez, M. A., Palomares-Navarro, J. J., & Ayala-Zavala, F. (2025). Virulence and Antibiotic Resistance of aEPEC/STEC Escherichia coli Pathotypes with Serotype Links to Shigella boydii 16 Isolated from Irrigation Water. Pathogens, 14(6), 549. https://doi.org/10.3390/pathogens14060549

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