Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea

Introduction The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. Aim In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. Methods To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. Results The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Conclusion Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, 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.


Introduction
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).
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 hemolyticuremic 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.
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, including VT1 and VT2 (STEC); bfpA and eaeA (EPEC); LT, STh and STp (ETEC); aggR (EAEC); ipaH (EIEC) (Lee et al., 2023a,b).

Genome sequencing, assembly, and annotation
Genomic DNA was extracted using the MagListo™ 5 M Genomic DNA Extraction Kit for cells and tissues (Bioneer, Daejeon, Korea) following the manufacturer's instructions.A DNA library was prepared using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA).Short-read sequencing was performed using the Illumina MiSeq sequencing system with the MiSeq Reagent Kit v3 (600-cycle).A hybrid genome assembly approach was employed to ensure a comprehensive genome sequence by integrating additional long-read sequence data obtained from PacBio Sequel (Pacific Bioscience, Menlo Park, CA, USA) or ONT MinION (Oxford Nanopore Technologies, Oxford, UK).A hybrid assembly of shortread sequence data (Illumina MiSeq) and long-read sequence data (PacBio Sequel, ONT MinION) was performed using Unicycler (v0.4.91 ; default options).The assembled genome was annotated using the Rapid Annotation with the Subsystem Technology toolkit, implemented in the BV-BRC annotation web service (v3.32.13a).

Prophage prediction and analysis
The PHAge Search Tool Enhanced Release (PHASTER) (Arndt et al., 2016) was used to detect bacteriophage sequences within distinct genome sequences.PHASTER predicted potential prophage regions, classifying them as "intact (scoring above 90), " "questionable (with scores ranging between 70 and 90), " or "incomplete (scoring below 70), " based on the presence of phage-related genes in the identified phage region of the genome containing Stx.Following the identification of these prophage sequences, the RAST toolkit within the BV-BRC genome annotation web service (v3.32.13a) was used for the annotation of virulence genes.

Identification of plasmid-associated sequences
PlasmidFinder 2.1 was used to investigate the plasmid features of the entire genome (Carattoli et al., 2014).The threshold for identification was 95% and the minimum coverage was set at 60%.Identification was based on the discovery of replicon sequences from various known plasmid incompatibility (Inc) groups, forming the basis for the identification process.Extracted plasmid sequences were annotated to detect virulence genes, including hemolysin-related genes, using Rapid Annotation with Subsystem Technology tools in the BV-BRC annotation web service (v3.32.13a).

Phylogenetic analysis and population structure analysis
We conducted a comparative genomic analysis of 32 hybrid STEC/aEPEC strains isolated in South Korea and 187 other pathogenic E. coli strains (Lee et al., 2023a,b).To analyze the pan-genome, we used the bacterial pan-genome analysis (BPGA) tool (v1.3) with default parameters.Clustering was performed using the USEARCH tool with 95% sequence identity as the cutoff value.A phylogenetic tree was constructed using the neighbor-joining method and visualized using the Interactive Tree of Life (iTOL) v6.For population structure analysis, RhierBAPs (Tonkin-Hill et al., 2018) were used.

Cell cytotoxicity and invasion assays
For cytotoxicity assessment, hybrid STEC/aEPEC strains were prepared as previously described (Roberts et al., 2001).The supernatants from 10 7 CFU of polymyxin B-treated bacteria were obtained and applied to the cell monolayer.Cell cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) release using the cytotoxicity detection kit (LDH) (Roche, Basel, Switzerland) according to the manufacturer's instructions.The absorbance was measured at 495 nm using a SpectraMax i3 platform (Molecular Devices, CA, USA).The results were calculated as relative values compared to those of untreated cells.The LDH release (% cytotoxicity) was calculated according to the following equation: ((LDH treated -LDH untreated )/(LDH totallysis − LDH untreated )) × 100.The experiments were performed in triplicate in duplicate wells.E. coli DH5α and the STEC strain ATCC 43890 were used as the non-cytotoxic control and cytotoxic positive control in the cytotoxicity assessment (Lang et al., 1994;Robinson et al., 2006).
To assess the invasive abilities of hybrid strains, a classical gentamicin protection assay was performed as previously described (Choi et al., 2007), with minor modifications.The cultures of hybrid STEC/aEPEC strains were applied to the cell monolayer at a multiplicity of infection (MOI) of 100.Invasion ability was determined as the percentage of the initial colony-forming units recovered after gentamicin treatment and expressed as relative invasion (%) of the non-invasive control strain, E. coli DH5α, defined as 100%.The experiments were performed twice in duplicate wells.E. coli DH5α and the EPEC strain E2348/69 were used as the non-invasive control and invasive positive control in the cell invasion assay (Rosa et al., 2001;Robinson et al., 2006;Iguchi et al., 2009).

Statistical analysis
The significance of differences between the bacterial strains was assessed using the GraphPad Prism 5.01 software, by using the Student's unpaired t-test.The data are represented as means and standard errors of the mean (SEM) from two independent experiments.

Genome assemblies of STEC/aEPEC hybrids
Thirty-two hybrid STEC/aEPEC strains from South Korea were sequenced; 16 strains had one chromosome and one plasmid, 11 had one chromosome and two plasmids, and five had one chromosome and three plasmids.Genomic characteristics of the hybrid STEC/ aEPEC strains are summarized in Table 2.The genome lengths of these strains ranged from 5,314,960 to 5,984,701 bp.In addition, these strains had G + C contents between 50.3 and 50.8%, the number of coding DNA sequences was between 5,437 and 9,866, tRNA genes between 87 and 105, and rRNA genes between 22 and 26.

Phage characterization
To detect the presence of stx-encoding phage in hybrid STEC/ aEPEC strains, we conducted a comprehensive identification of bacteriophage sequences using PHASTER.The outcomes of our investigation, involving 32 hybrid STEC/aEPEC strains, are presented in Table 2. Validation of the entire set of stx1 gene sequences was confirmed within the respective "intact" phage sequence region.Notably, the stx1 (stx 1a and stx 1b) gene sequence was detected in most STEC/aEPEC strains (30/32), with only two strains (MFDS1009661 and MFDS1019524) confirmed to possess both stx1 (stx 1a and stx 1b) and stx2 (stx 2a and stx 2b) gene sequences.

Phylogenetic analysis and population structure analysis
This study aimed to elucidate the genomic relationships between STEC/aEPEC hybrids and other pathogenic E. coli strains.The genomes of 187 isolates, comprising 41 STEC, 46 ETEC, 72 EPEC, 18 EAEC, and 10 EIEC strains, were used for the phylogenetic analysis to determine the genomic relationship between the STEC/aEPEC hybrids and other pathogenic E. coli isolates.The 187 genome datasets included the sequencing results of 160 pathogenic E. coli as well as 32 hybrid STEC/ aEPEC genomes, which were deposited in the NCBI database.The genomes analyzed in this study are summarized in Supplementary Table S3.The phylogenetic analysis based on BPGA, which used coding sequences (CDSs) with protein sequences, revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of STEC virulence genes during their emergence (Figure 5A).Furthermore, hierBAPS analysis was based on aligning SNPs from pan-genomes, and it's utilized to identify genomic similarities among strains through hierarchical clustering of DNA sequence data.The phylogenetic analysis based on hierBAPS revealed their division into six primary sequence clusters (Bayesian analysis of population structure [BAPS] hierarchical level 1), which were further segregated into 28 lineages (BAPS level 2) (Figure 5B).All 32 hybrid strains were closely related to EPEC and were distributed across six groups (two at level 1 and six at level 2) within the total distribution.

Discussion
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 The heatmap of the protein-encoding genes within the LEE region of STEC strains (RM10042 and RM10466), EPEC strains (E2348/69 and E110019), non-O157 EHEC strain (11,368 and 12,009), and STEC/EPEC genomes analyzed in this study.Heatmap showing the presence/absence of proteinencoding genes (y-axis) within hybrid STEC/aEPEC isolates identified in this study (x-axis).The presence of protein-encoding genes within the LEE region is shown in blue, whereas the absence is shown in white, as indicated in the color key.Heatmap produced using the gplot (v3.1.3)package in R (v4.1.3). 10.3389/fmicb.2024.1398262Frontiers in Microbiology 09 frontiersin.organd 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, Comparative analysis of hemolysin-encoding plasmids.The pangenome shows the hemolysin-encoding plasmids for the 32 hybrid STEC/aEPEC strains.This pangenome is divided into plasmids IncFI1, IncFIB, IncY, and phage.The graphical circular maps of plasmids were generated using GView.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 phageencoded 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 Heatmap of antimicrobial profiles across hybrid STEC/aEPEC strains.Heatmap showing the presence/absence of antimicrobial resistance (y-axis) among the hybrid STEC/aEPEC isolates identified in this study (x-axis).The presence of antimicrobial resistance genes is shown in purple, and the absence in white.Heatmap was generated using the gplot (v3.1.3)package in R software (v4.1.3).

FIGURE 5
Phylogenetic and population structure analysis of hybrid STEC/aEPEC strains.(A) Green bars represent the 32 hybrid STEC/aEPEC strains that are closely related to specific EPEC strains.(B) Sequence clusters (1 to 6) are indicated in the outer colored dot, which are further divided into 28 lineages (inner ring).The green bar represents 32 STEC/EPEC hybrid strains isolated identified in this study.Among the identified strains, 32 hybrid strains closely related to EPEC were divided into six groups (two level 1, six level 2).
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.

Conclusion
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.

FIGURE 6
FIGURE 6 Assessment of cytotoxicity and invasive ability of STEC/aEPEC strains.(A) Cytotoxicity (%) of STEC/aEPEC hybrid strains was measured via LDH release from HeLa cells.Error bars represent S.E.M. from two independent experiments.ATCC 43890 and E2348_69 were used as reference strains for STEC and EPEC, respectively.N.D., Not detected.(B) The invasive ability of STEC/aEPEC hybrid strains was expressed as the relative invasion (%) of the negative control strain E. coli DH5α, defined as 100%.Error bars represent S.E.M. from two independent experiments.Statistical analyses were performed using Student's t-test.*p < 0.05, **p < 0.01, ***p < =0.005.