Escherichia coli (E. coli) commonly resides in the gastrointestinal tracts of humans and animals as part of their natural gut flora (Kaper et al., 2004). Although most strains are harmless members of this gut community, certain E. coli variants, including those that have acquired specific virulence traits, can cause diarrheal and extraintestinal illnesses in humans and animals (Kaper et al., 2004; Dobrindt, 2005). Diarrheagenic E. coli (DEC) accounts for approximately 30–40% of acute diarrhea cases in children under 5 years old in developing countries (Dutta et al., 2013; Miliwebsky et al., 2016) and is a significant cause of both isolated instances and outbreaks of diarrhea worldwide (Croxen et al., 2013).

DEC, characterized by distinct combinations of virulence characteristics, are categorized into pathotypes, each with specific host preferences, global occurrence, disease impacts, and transmission methods (Croxen and Finlay, 2010). DEC are categorized into six main pathotypes: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC). Each E. coli pathotype possesses distinct pathogenic mechanisms and a unique set of virulence factors encoded by specific gene clusters (Jesser and Levy, 2020; Pakbin et al., 2021). These genes related to pathogenicity may control functions such as adhesion, invasion, attachment, iron acquisition, motility, and toxin activity (Kaper et al., 2004; Croxen and Finlay, 2010).

STEC is an E. coli pathotype capable of producing at least one of the Shiga toxins (Stxs), namely Stx1 and Stx2 (Kaper et al., 2004; Croxen et al., 2013). Within each group, numerous subtypes or variants of Stx have been identified and characterized (Burgos and Beutin, 2012). STEC is a globally prevalent pathogen associated with human diseases that primarily causes diarrhea, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS) (Jesser and Levy, 2020; Pakbin et al., 2021). EPEC induces diarrhea and initiates the formation of attaching and effacing (A/E) lesions in the human gut epithelium (Kaper et al., 2004; Croxen et al., 2013). EPEC strains are categorized as either typical or atypical according to whether or not they have the E. coli adherence factor plasmid (EAF) expressing the bfpA gene. This gene is responsible for the production of bundle-forming pili. Typical EPEC (tEPEC) possesses both the locus of enterocyte effacement (LEE) region responsible for attaching and causing effacing lesions (eae), and bfpA. In contrast, atypical EPEC (aEPEC) lacks bfpA (Jesser and Levy, 2020; Pakbin et al., 2021). aEPEC strains exhibit comparable prevalence rates among healthy adults and children with diarrhea, and are also found in asymptomatic adults (Hazen et al., 2016). However, certain aEPEC strains have been linked to diarrhea outbreaks (Vieira et al., 2016).

The Enterohemorrhagic Escherichia coli (EHEC) is commonly used to refer to a subset of STEC strains that simultaneously harbors genes for Stx production and LEE. As STEC strains have the potential to induce diarrhea and hemolytic uremic syndrome (HUS) in human subjects regardless of the presence of LEE, this PAI is an accessory set of virulence genes that enhances STEC pathogenicity. EHEC are considered a pathotype of E. coli and are referred to as such in the scientific literature rather than referring to them as hybrid STEC/aEPEC. However, according to previous studies, there are reports indicating that EPEC/STEC hetero-pathogens consist of atypical EPEC carrying Stx (Croxen et al., 2013; Eichhorn et al., 2015; Gomes et al., 2016; Silva et al., 2019). Especially, Gioia-Di Chiacchio et al. (2018) were the first to report a typical EPEC strain carrying Stx, identified in eight E. coli strains isolated from birds, which simultaneously harbored Stx2, LEE, and BFP.

Globally, emerging hybrid diarrheagenic E. coli strains incorporating genetic markers from diverse pathotypes pose substantial public health concerns (Santos et al., 2020). This emergence is attributed to the horizontal gene transfer (HGT) among diarrheagenic E. coli or the prevalence of E. coli virulence genes typically located on plasmids, enabling transmission via conjugation (Kaper et al., 2004; Dobrindt, 2005; Geurtsen et al., 2022). Hybrid DEC pathotype strains are increasingly reported worldwide. Different hybrid strains, such as EPEC/ETEC, have been identified in India (Dutta et al., 2015), with Germany (Bielaszewska et al., 2011) and Indonesia (Setyarini et al., 2020) reporting STEC/EAEC hybrids. Brazil has documented cases of EPEC/STEC (Gioia-Di Chiacchio et al., 2018) and EPEC/EAEC (de Lira et al., 2021). Sweden has several instances of STEC/ETEC (Bai et al., 2019). Diarrheal disease outbreaks associated with various DEC pathotypes occurred in Germany in 2011 (Buchholz et al., 2011), Japan in 2016 (Furukawa et al., 2018) and 2020 (Kashima et al., 2021), Nottingham, UK in 2014 (Newitt et al., 2016), South Korea in 2018 (Lim et al., 2020), and the United Kingdom in 2020 (Mulchandani et al., 2021). Hybrid strains are characterized by the presence of virulence genes unique to multiple E. coli pathotypes, typically detected via PCR or comprehensive whole-genome sequencing (WGS) (Jesser and Levy, 2020; Lee et al., 2023a,b). Cell-based assays have also been used to assess the virulence potential of hybrid strains (Valiatti et al., 2020).

The purpose of the present study was to characterize the genetic structure and evaluate the virulence potential of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea. This study enhances our understanding of the horizontal transfer of several virulence-related genes via mobile genetic elements (MGEs) and provides information on the virulence potential of hybrid STEC/aEPEC strains. Based on our findings, we highlighted the potential implications of these hybrid E. coli strains for public health, emphasizing the need for comprehensive monitoring to mitigate risks associated with the horizontal transfer of virulence-related genes.

STEC and EPEC, both foodborne pathogens, can cause severe consequences such as potentially fatal infant diarrhea and hemolytic uremic syndrome, respectively. As STEC strains have the potential to induce diarrhea and hemolytic uremic syndrome (HUS) in human subjects regardless of the presence of LEE, this PAI is an accessory set of virulence genes that enhances STEC pathogenicity. The STEC/EPEC pathotype has been linked to numerous global outbreaks, severe symptoms, and, in some cases, fatal outcomes. Here, we first report 32 hybrid STEC/aEPEC strains identified in South Korea between 2015 and 2021, aiming to profile their virulence and associated genes for deeper exploration of their phylogenetic relationships with other pathogenic E. coli strains. Additionally, the genotypic profiles of AMR were analyzed. Furthermore, a cell-based assay was used to evaluate the virulence potential of hybrid STEC/aEPEC strains.

To molecularly characterize all hybrid STEC/aEPEC strains, a combination of real-time PCR and serotyping was used. WGS analysis of these hybrids corroborated the findings related to the presence of virulence factors, serotyping, and antimicrobial susceptibility. All 32 STEC/EPEC hybrid strains confirmed the presence of genes encoding Shiga toxin 1 and/or 2 (stx1a, stx1b and/or stx2a, stx2b) and E. coli attaching and effacing lesions (eae). Stxs, the major virulence factors of STEC, are broadly classified into two types: stx1 and stx2. Furthermore, in the Stx1 group, stx1a is found in major STEC strains causing HC and HUS in humans, whereas stx1c and stx1d are rarely associated with human infections. In the Stx2 group, stx2a, stx2c, and stx2d were reported to be more virulent in human infections compared to isolates carrying stx2b, stx2e, stx2f, and stx2g (Burgos and Beutin, 2012; Probert et al., 2014; Zhi et al., 2019; Bai et al., 2021). Epidemiological data indicate a stronger association between Stx2-producing E. coli strains and HUS than strains producing Stx1 (Basu et al., 2016). However, the E. coli O157:H7 strain, producing stx1, and not stx2, was isolated from a Korean patient with HUS (Kim et al., 1998). Most hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea harbored stx1. EPEC strains were categorized as typical or atypical based on the presence or absence of the E. coli adherence factor plasmid (EAF) housing bfpA, which encodes the bundle-forming pili (BFP). tEPEC includes the LEE region, including eaeA for attaching and effacing lesions, and bfpA. In contrast, aEPEC lacks bfpA. aEPEC causes endemic, epidemic, and persistent diarrhea in both humans and farm animals (Trabulsi et al., 2002), with certain cases linked to acute illness in humans (Abba et al., 2009). Our investigation confirmed that the STEC/EPEC hybrid strains identified in South Korea belonged to the aEPEC category and were distinguished by the absence of a gene encoding BFP.

All hybrid STEC/aEPEC strains encompassed chromosomally encoded virulence genes characteristic of EPEC, such as intimin and TTSS within the LEE region, and additional effectors not linked to LEE. The hallmark of an EPEC infection is the development of A/E lesions within the intestinal epithelium (Cepeda-Molero et al., 2017). These lesions result from genes situated on the LEE pathogenicity island, which encodes adhesin intimin, a TTSS, and six effectors, including the pivotal translocated intimin receptor (Tir) (Cepeda-Molero et al., 2017). In additionally, the chromosome of STEC/aEPEC contains T6SS which along with TTSS are important for cell–cell interaction and virulence factors. Previous studies have shown that the prevalence of the T6SS gene cluster is significantly higher in pathogenic E. coli strains compared to non-pathogenic E. coli strains. Pathogenic strains of E. coli have been identified with multiple T6SSs, some carrying more than one T6SS cluster, while others possess a single T6SS cluster but with multiple copies of hcp and vgrG genes. This highlights the functional role of the T6SS system in enhancing host pathogenicity.

The hybrid STEC/aEPEC strains in South Korea carry a phage-encoded STEC virulence gene (stx) responsible for Stx production. Additionally, phylogenetic tree analysis revealed close relationships between all hybrids and specific EPEC strains. These findings indicated that STEC/aEPEC hybrids potentially originated from EPEC strains that acquired STEC virulence genes via phage transmission. Moreover, our investigation revealed that all the hybrid strains possessed plasmid-carried enterohemolysin genes (ehxCABD). Enterohemolysin (Ehx) is a pore-forming toxin in enterohemorrhagic Escherichia coli that increases virulence in various clinical infections and associated with the presence of shiga toxin (Schwidder et al., 2019). This toxin is encoded on plasmids as part of an operon comprised of four genes: ehxA (coding for functional toxin), ehxC (managing post-translational modification of EhxA), ehxB and ehxD (responsible for the transport of EhxA through the inner membrane) (Kolenda et al., 2021).

The hybrids represented diverse serotypes [O103:H2 (n = 13), O26:H11 (n = 3), O74:H25 (n = 3), O84:H2 (n = 3), O108:H25 (n = 3), O98:H21 (n = 2), O103:H8 (n = 2), O71:H8 (n = 1), O156:H25 (n = 1), and O177:H25 (n = 1)]. The O157:H7 serotype strains are the primary human pathogens among enterohemorrhagic E. coli (EHEC) strains; however, other non-O157 serotypes (O26, O91, O103, O111, O121, and O145) have also exhibited the capacity to cause severe illnesses (Allerberger et al., 2003; Karmali et al., 2003; Kaper et al., 2004; Johnson et al., 2006; Messens et al., 2015; Soysal et al., 2016). Moreover, incidents of EHEC O80:H2 triggering lethal complications in HUS have recently emerged in France (Mariani-Kurkdjian et al., 2014; Soysal et al., 2016), the Netherlands (Wijnsma et al., 2017), and Switzerland (Nüesch-Inderbinen et al., 2018).

The cytotoxicity of Shiga toxin-producing E. coli is linked to the presence of stx (Maldonado et al., 2005). Polymyxin B is effective in extracting intracellular proteins, including heat-labile enterotoxins and Shiga-like toxins, from the periplasmic space of gram-negative bacteria (Evans et al., 1974; Karmali et al., 1985). Using the Polymyxin B extraction method for bacterial preparations, we examined the toxic potential of Stxs in STEC/aEPEC hybrids. All hybrid strains harboring stx and the STEC reference strain ATCC 43890 were cytotoxic to HeLa cells, whereas the EPEC reference strain E2348/69 and non-cytotoxic control E. coli DH5α showed no cytotoxicity. Notably, hybrid strains (MFDS1009661 and MFDS1019524) harboring both stx1 and stx2 were highly cytotoxic (45%) (Table 2), without discernible differences in cytotoxicity from the STEC strain ATCC 43890 (p > 0.05, Student’s t-test). Some hybrid strains lacking the stx2 gene sequence also exhibited high cytotoxicity to HeLa cells, suggesting the involvement of other unidentified virulence genes in the cytotoxicity of hybrid STEC/aEPEC strains.

EPEC adherence occurs through three stages of adherence to epithelial cells: initial adherence (i), signal transduction (ii), and intimate attachment (iii) (Donnenberg et al., 1997). In particular, the EPEC adherence factor (EAF) plasmid confers localized adherence on the epithelial cells called the attaching and effacing (A/E) lesions by expressing BFP, which makes differentiation between tEPEC and aEPEC (Nataro and Kaper, 1998; Trabulsi et al., 2002). It is also distinct from the invasion mechanism of enteroinvasive E. coli (EIEC), which is mediated by the invasion plasmid (pINV) encoding genetic determinants for epithelial cell invasion, including the invasion plasmid antigen gene H (ipaH) (Sethabutr et al., 1993). The results of virulence gene mapping for 32 STEC/aEPEC strains demonstrated that ipaH sequences were not detected in the hybrid genomes (Figure 1). Considering the lack of EAF plasmid in aEPEC strains, the absence of ipaH suggests that the invasive potential of STEC/aEPEC hybrids may be derived from mechanisms different from those of tEPEC or EIEC. Instead, it was reported that some aEPEC strains possessing the intimin-encoding gene (eae) exhibited invasive potential in human intestinal cells in vitro with varying invasion efficiencies (Hernandes et al., 2008; Yamamoto et al., 2009). Despite the absence of the genes responsible for tEPEC and EIEC invasion, the presence of intimin within the genomes of 32 STEC/aEPEC hybrids may support the possibility of highly invasive strains. However, further analyses are still needed to evaluate the ability of hybrid strains to invade the epithelial cells in vivo. As the STEC strains are inefficient in invading epithelial cells (Oelschlaeger et al., 1994), invasive ability distinguishes EPEC from STEC types. The five hybrid strains were highly invasive to HeLa cells compared to the EPEC strain E2348/69. Notably, all five hybrid strains were related to EPEC in the phylogenetic analysis, suggesting a correlation between the phylogenetic and virulence profiles of the STEC/aEPEC hybrids. Moreover, all ST 1034 (n = 11) hybrid STEC/aEPEC strains displayed higher invasive potential than the EPEC strain E2348/69, including the highly invasive and cytotoxic hybrid strains MFDS1017062 and MFDS1021098. In particular, these genomes harbored two T6SS gene clusters, each containing the 13 core T6SS genes (TssJ, TssL, TssM, TssK, TssF, TssG, TssE, TssA, TssB, TssC, ClpV, Hcp, VgrG) necessary for the proper functioning of a T6SS. These results suggested the need to explore possible correlations between STs and virulence profiles in hybrid E. coli strains.

This study investigated the transfer of virulence genes in hybrid STEC/aEPEC strains identified in South Korea, emphasizing the efficacy of WGS as a robust method for scrutinizing bacterial genomes, particularly in detecting regions containing MGEs like phages and plasmids. Antibiotic resistance was confirmed in only three hybrid STEC/aEPEC strains (MFDS1011030, MFDS1012302, and MFDS1021098), displaying multiple drug resistance. Moreover, five hybrid strains (MFDS1006809, MFDS1008632, MFDS1009661, MFDS1012302, and MFDS1012330) showed cytotoxic activity against HeLa cells and significantly higher invasive ability than the EPEC strain E2348/69. These genomic and virulence findings substantially contribute to our understanding of hybrid STEC/aEPEC strains and pave the way for further investigations of E. coli pathogenicity. Furthermore, this study observes the emergence of MDR hybrid E. coli strains with high virulence potential.

This study documented the virulence markers and antimicrobial resistance profiles of hybrid STEC/aEPEC strains isolated in South Korea. Comprehensive genome-wide phylogenetic analysis demonstrated close genetic relationships between all hybrids and specific EPEC strains. Furthermore, the genes encoding stx1 or stx2 within these hybrid strains were harbored by phages. Additionally, some hybrid strains exhibited cytotoxicity and invasive abilities in human epithelial HeLa cells. Notably, all hybrid STEC/aEPEC strains that belonged to ST 1034 (n = 11) had a higher invasive potential than the EPEC strain E2348/69, including the two highly invasive and highly cytotoxic hybrid strains MFDS1017062 and MFDS1021098. Our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone. Furthermore, these genomic and virulence findings considerably contribute to our understanding of hybrid STEC/aEPEC strains and encourage further investigations into E. coli pathogenicity.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

WL: Data curation, Methodology, Writing – original draft. JH: Data curation, Methodology, Writing – review & editing. JC: Data curation, Methodology, Writing – review & editing. YJ: Data curation, Methodology, Writing – review & editing. EK: Methodology, Writing – review & editing. EA: Methodology, Writing – review & editing. SeK: Writing – review & editing. HS: Writing – review & editing. SR: Writing – review & editing. SoK: Conceptualization, Supervision, Writing – review & editing. H-YK: Supervision, Writing – review & editing.