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Article

Genome-Based Characterization of Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains Isolated in South Korea, 2016–2020

by
Woojung Lee
1,2,
Min-Hee Kim
1,
Soohyun Sung
1,
Eiseul Kim
2,
Eun Sook An
1,
Seung Hwan Kim
1,
Soon Han Kim
1,* and
Hae-Yeong Kim
2,*
1
Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea
2
Institute of Life Sciences & Resources, Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea
*
Authors to whom correspondence should be addressed.
Microorganisms 2023, 11(5), 1285; https://doi.org/10.3390/microorganisms11051285
Submission received: 5 April 2023 / Revised: 11 May 2023 / Accepted: 11 May 2023 / Published: 15 May 2023
(This article belongs to the Special Issue Escherichia coli and Food Safety 2.0)
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Abstract

:
The global emergence of hybrid diarrheagenic E. coli strains incorporating genetic markers from different pathotypes is a public health concern. Hybrids of Shiga toxin-producing and enterotoxigenic E. coli (STEC/ETEC) are associated with diarrhea and hemolytic uremic syndrome (HUS) in humans. In this study, we identified and characterized STEC/ETEC hybrid strains isolated from livestock feces (cattle and pigs) and animal food sources (beef, pork, and meat patties) in South Korea between 2016 and 2020. The strains were positive for genes from STEC and ETEC, such as stx (encodes Shiga toxins, Stxs) and est (encodes heat-stable enterotoxins, ST), respectively. The strains belong to diverse serogroups (O100, O168, O8, O155, O2, O141, O148, and O174) and sequence types (ST446, ST1021, ST21, ST74, ST785, ST670, ST1780, ST1782, ST10, and ST726). Genome-wide phylogenetic analysis revealed that these hybrids were closely related to certain ETEC and STEC strains, implying the potential acquisition of Stx-phage and/or ETEC virulence genes during the emergence of STEC/ETEC hybrids. Particularly, STEC/ETEC strains isolated from livestock feces and animal source foods mostly exhibited close relatedness with ETEC strains. These findings allow further exploration of the pathogenicity and virulence of STEC/ETEC hybrid strains and may serve as a data source for future comparative studies in evolutionary biology.

1. Introduction

Escherichia coli is commonly regarded as a nonpathogenic beneficial inhabitant of the gastrointestinal tract. However, several pathogenic strains have acquired specific virulence factors that are responsible for various intestinal and extraintestinal diseases, including diarrhea, acute inflammation, hemorrhagic colitis, urinary tract infections, septicemia, and neonatal meningitis. Diarrheagenic Escherichia coli (DEC) causes 30–40% of acute diarrhea episodes in children <5 years in developing countries [1]. According to the WHO Global Burden of Foodborne Diseases report, >300 million illnesses and nearly 200,000 deaths are caused by DEC globally each year [2]. Major diarrheagenic E. coli (DEC) strains are subdivided into several pathotypes based on the presence of specific virulence traits directly related to disease development [3,4,5,6]. The DEC pathotypes include enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC). Many of these pathotypes are foodborne pathogens that raise public health concerns and cause several outbreaks in industrialized and developing countries [7,8,9].
STEC and ETEC are major causes of diarrhea in humans and animals worldwide. STEC is characterized by the presence of the Shiga toxin 1 or 2 genes (stx1 or stx2), which are generally acquired by a lambda-like bacteriophage [10]. Shiga toxins 1 and 2 (Stx1 and Stx2, respectively) differ in their virulence and host specificity, with Stx2 being most commonly associated with severe illnesses (hemolytic uremic syndrome (HUS), hospitalization, and bloody diarrhea) in humans [11,12]. ETEC is characterized by its ability to produce either a heat-labile (LT) or heat-stable (ST) enterotoxin and carries a diverse set of colonization factors (CFs) for adherence to the intestinal epithelium [13]. It is a major cause of diarrhea among children living in and tourists traveling to developing countries.
Hybrid DEC strains that combine genetic markers belonging to different pathotypes have emerged worldwide and are a public health concern [14]. Numerous virulence markers are frequently carried on mobile genetic elements (MGEs), such as phages and plasmids, allowing the transmission of virulence genes via horizontal gene transfer, leading to the emergence of hybrid pathotypes [3,15,16,17,18]. Hybrid E. coli strains comprising genetic markers of different pathotypes have been identified owing to the technological advances that provide a better understanding of the genomic and virulence mechanisms of DEC [19].
The most well-documented example is the E. coli O104:H4 strain, which caused a severe outbreak of acute gastroenteritis and HUS in Germany in 2011 [20]. This strain produced Stx2, a signature feature of the STEC pathotype, and it carried a plasmid containing the genes encoding aggregative adherence fimbriae (AAF), which mediate aggregative adherence in EAEC [21,22,23]. Furthermore, hybrids of STEC and ETEC strains (STEC/ETEC) have been recently reported in various countries, including Bangladesh, Sweden, and South Korea, some of which have been associated with diarrheal diseases and HUS in humans [24,25,26,27,28].
Few studies have reported the virulence and antibiotic resistance profiles of STEC/ETEC hybrid strains isolated from livestock feces (cattle and pigs) and animal source foods (beef, pork, and meat patties) in South Korea. This study investigated the genomes of STEC/ETEC hybrid strains to identify the virulence and antibiotic resistance genes they harbored and to determine their phylogenetic position among other E. coli strains. The genomic properties of these strains were investigated via real-time PCR and whole-genome sequencing (WGS). Phylogenetic analysis was performed to assess their phylogeny in a collection of E. coli strains from diverse pathotypes. Based on our findings, we addressed the potential importance of these hybrid E. coli strains for public health.

2. Materials and Methods

2.1. Bacterial Strains and Serotyping

Pathogenic E. coli strains that originated from the Korean Culture Collection for Foodborne Pathogens (Ministry of Food and Drug Safety) were identified. All 1025 pathogenic E. coli strains isolated in South Korea between 2016 and 2020 were analyzed. Twenty-seven hybrid Shigatoxigenic and enterotoxigenic Escherichia coli (STEC/ETEC) strains were isolated from livestock feces (cattle and pigs) and animal source foods (beef, pork, and meat patties). The strains selected for this study are listed in Table 1. Typical E. coli colonies (blue-green color) on 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (BCIG) agar (Oxoid, UK) were sub-cultured on Tryptic Soy Agar (Oxoid, UK) and then incubated at 37 °C for 18–24 h. The isolates were identified using VITEK MS (BioMerieux Inc., Marcy-l’Etoile, France). The serotype was determined by the agglutination of the bacteria with specific somatic (O1 to O181) antisera [Laboratorio de Referencia de E. coli (LREC), Lugo, Spain] to identify variants of the somatic (O) antigens [29,30,31].

2.2. Antimicrobial Susceptibility Tests

Antimicrobial susceptibility tests were performed using Sensititre KRN6F panels (Trek Diagnostic Systems, Cleveland, OH, USA) following the manufacturer’s instructions. The antimicrobial susceptibility of the isolated strains was determined using the 16 antimicrobials described as follows: amoxicillin–clavulanic acid, ampicillin, cefoxitin, cefotaxime, ceftazidime, cefepime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole. The MIC (minimum inhibitory concentration) value of these antimicrobials was determined with the microbroth dilution method. The Clinical and Laboratory Standards Institute guidelines and the U.S. National Antimicrobial Resistance Monitoring System were used to interpret susceptibility results expressed as MICs. For these agents, the degree of increase in resistance was determined by referring to the resistance level of the standard strain, ATCC 25922.

2.3. Real-Time PCR-Based Identification of Hybrid Strains

DNA was extracted from the bacterial cultures using automated equipment (EZ1 Advanced XL, Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. The extracted DNA was used as a template for real-time PCR, which was performed using a PowerCheckTM 20/15 Pathogen Multiplex Real-time PCR kit (Kogene Biotech Co., Ltd., Seoul, Korea) to detect virulence genes. Amplification was performed using an ABI 7500 Fast Real-time PCR system (Applied Biosystems, Waltham, MA, USA) at 50 °C for 2 min for 1 cycle, 95 °C for 10 min for 1 cycle, followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min. The following genes from different DEC pathogens were detected: VT1 and VT2 (STEC); bfpA and eaeA (EPEC); LT, STh, and STp (ETEC); aggR (EAEC); ipaH (EIEC).

2.4. Genome Sequencing, Assembly, and Annotation

Genomic DNA was extracted using the MagListoTM 5M Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol. DNA integrity and concentration were determined using standard agarose gel electrophoresis and a QubitTM 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), respectively. A DNA library was prepared using a Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA). Sequencing was performed using a MiSeq sequencing system (Illumina) and MiSeq Reagent Kit v3 (600-cycle) (Illumina). The contigs (FASTQ sequence files) were assembled de novo using the CLC Workbench (version 12.0; Qiagen, Hilden, Germany). To obtain high-quality data and determine the complete genomic sequence, the hybrid genome was assembled using additional long-read sequence data obtained from PacBio Sequel (Pacific Bioscience, Menlo Park, CA, USA). Hybrid assembly of raw FASTQ PacBio sequence long-read sequence data and Illumina MiSeq short-read FASTQ sequence data was performed using Unicycler (v0.4.9; https://github.com/rrwick/Unicycler (accessed on 1 January 2023); default settings). The assembled genome was annotated using the Rapid Annotation using Subsystem Technology (RAST) toolkit in the PATRIC genome annotation web service (v3.6.12).

2.5. DNA Sequence and Bioinformatics Analysis

Virulence factors and antimicrobial resistance genes were identified using the virulence factor database and ResFinder v4.1 [32,33], respectively. Adhesion and colonization factors in the human intestine required for STEC pathogenesis, such as the locus of adhesion and autoaggregation (LAA)-related genes, were identified using the Basic Local Alignment Search Tool (BLAST). Mobile genetic elements were identified using MobileElementFinder (https://cge.cbs.dtu.dk/services/MobileElementFinder/) (accessed on 22 February 2023) [34]. CRISPRCasFinder (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) (accessed on 23 April 2023) was used to analyze the CRISPRs and cas genes [35]. Whole-genome multilocus sequence typing (wgMLST) was performed with BioNumerics (v8.0; Applied Maths, Sint-Martens-Latem, Belgium), where 17,380 loci from E. coli and Shigella were included for analysis. In addition, the web-based serotyping tool, SerotypeFinder 2.0 [36], was used to predict the antigen profiles of E. coli strains.

2.6. Prophage Prediction and Analysis

Bacteriophage sequences within the unique genome sequence were identified using PHAge Search Tool Enhanced Release (PHASTER) [37]. PHASTER was used to predict putative prophage regions as “intact (score > 90),” “questionable (score 70–90),” or “incomplete (score < 70)” based on the proportion of phage-related genes in the identified phage region of the assembled hybrid genome. The extracted prophage sequences were annotated to identify virulence genes using the RAST toolkit in the PATRIC genome annotation web service (v3.6.12).

2.7. Identification of Plasmid-Associated Sequences

Plasmid features of the assembled hybrid genomes were analyzed using PlasmidFinder 2.1 [38]. The threshold and minimum coverage for identification were set to 95% and 60%, respectively. The identification was based on the detection of replicon sequences belonging to several known plasmid incompatibility (Inc) groups. The extracted plasmid sequences were annotated to identify virulence genes using the RAST toolkit in the PATRIC genome annotation web service (v3.6.12).

2.8. Phylogenetic Analysis and Population Structure Analysis

Comparative genomic analysis was performed on 27 STEC/ETEC hybrid strains isolated from livestock feces (cattle, pigs) and animal source foods (beef, pork, meat patties) in South Korea and 187 pathogenic E. coli strains. The genomic sequences of 160 strains isolated from food and the environment in South Korea and 27 other pathogenic E. coli strains are available at the National Center for Biotechnology Information (NCBI). The genomes analyzed in this study are summarized in Supplementary Table S5. The pan-genome was analyzed using the bacterial pan-genome analysis (BPGA) tool (v1.3; default parameters). The USEARCH tool was used for clustering with 95% sequence identity as the cut-off value. The phylogenetic tree was clustered using the neighbor-joining method and visualized using the Interactive Tree of Life (iTOL) v6. The population structure analysis was performed using RhierBAPs [39].

3. Results

3.1. Genome Assemblies of STEC/ETEC Hybrids

Twenty-seven STEC/ETEC hybrid strains isolated from livestock feces and animal source foods in South Korea were sequenced. All strains had one chromosome and one plasmid. The genomic characteristics of the hybrid STEC/ETEC strains are summarized in Table 1. The genome lengths of these isolates ranged from 5,064,469 to 5,865,149 bp, with coverage ranging from 126× to 524×. In addition, the G + C content of the genomes of these strains was between 50.3% and 50.9%, the length of the coding DNA sequences (CDSs) was between 5081 and 6141 bp, and the number of tRNA and rRNA genes was 82–107 and 22, respectively.

3.2. In Silico Identification of Virulence and CRISPR-Associated (Cas) Genes

The initial screening of pathogenic E. coli strains was conducted using real-time PCR. The hybrid STEC/ETEC isolates harbored both Shiga toxin 2 (stx2) and heat-stable enterotoxins (est) encoding genes. Subsequently, we performed virulence gene mapping to identify the various virulence factors present in the STEC/ETEC hybrid genomes (Figure 1). Multiple virulence factors have been implicated in E. coli pathogenesis, including the Shiga toxin and enterotoxins, as well as other factors such as adhesion factors, colonization factors (CFs), non-LEE-encoded TTSS effectors, and secretion systems. Detailed results of virulence gene mapping of these hybrid genomes are shown in Supplementary Table S1. Importantly, we also detected genes encoding LAA, such as hes, iha, lesP, and agn43, which are related to STEC pathogenicity. The most prevalent gene was iha (59.3%), followed by agn43, hes, and lesP that were present in 25.9, 18.5, and 11.1% of the hybrid strains, respectively. In addition, the CRISPRFinder server identified a type I CRISPR/Cas system in all hybrid strains. Additionally, most of the STEC/ETEC hybrid strains (26/27) identified the type I-E system. All hybrid strains harbored the cas3 gene, which is the signature of type I CRISPR/Cas systems, responsible for target DNA cleavage and degradation [40]. Furthermore, most of the STEC/ETEC hybrid strains (26/27) harbored the cas1, cas2, cas5, cas6, and cas7 genes as well as the cas3 gene.

3.3. In Silico Identification of Antimicrobial Resistance Genes

ResFinder was used to predict antimicrobial resistance genes in the hybrid genomes (Figure 2). Most of the genomes (16/27) contained at least two antibiotic (ampicillin, piperacillin, streptomycin, and ticarcillin) resistance genes, whereas nine isolates were negative for them. The comprehensive results of the antimicrobial resistance gene mapping of the hybrid E. coli genomes are shown in Supplementary Table S2. High rates of resistance gene were observed for tetracycline (55.6%), doxycycline (55.6%), chloramphenicol (51.9%), sulfamethoxazole (51.9%), florfenicol (48.1%), streptomycin (48.1%), amoxicillin (44.4%), ampicillin (44.4%), and piperacillin (44.4%). Especially, the tetA and tetB genes were found at the highest frequency in hybrid E. coli strains. We additionally performed antimicrobial susceptibility tests to determine the phenotypic profile of antimicrobial resistance in STEC/ETEC hybrid strains. The phenotypic profile of antimicrobial resistance is described in detail in Supplementary Table S3. Comparing the WGS-based AMR genotype to the antimicrobial susceptibility testing-based phenotype for 13 antibiotics (ampicillin, cefepime, cefoxitin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, colistin, nalidixic acid, meropenem, gentamicin, streptomycin, tetracycline) revealed concordant results for 22 of the 27 STEC/ETEC hybrid strains (81.5%). In five STEC/ETEC hybrid strains, only antibiotic resistance genes were identified, but no phenotypes. Although WGS can provide more information about isolates, genomic approaches cannot always predict phenotypes because the level of gene expression and protein production from identified genes may differ between strains. Bacteria have gene-silencing mechanisms, and mutations may generate stop codons in the data [41].

3.4. Serotyping and Sequence Types of the Hybrids

The serotype and sequence type results for the 27 hybrid STEC/ETEC isolates are summarized in Table 2. Based on in silico serotyping, the 27 hybrid E. coli strains belonged to eight distinct O:H serogroups [O100:H30 (n = 7), O8:H9 (n = 5), O168:H8 (n = 5), O155:H21 (n = 4), O2:H25 (n = 3), O141:H29 (n = 1), O148:H7 (n = 1), and O174:H2 (n = 1)]. Especially, the O100:H30 serogroup was found at the highest frequency in hybrid E. coli strains. The hybrid strains represented diverse sequence types [ST446 (n = 7), ST1021 (n = 5), ST21 (n = 4), ST74 (n = 4), ST785 (n = 1), ST670 (n = 2), ST1780 (n = 1), ST1782 (n = 1), ST10 (n = 1), and ST726 (n = 1)].

3.5. Phage Characterization

To investigate the phage-mediated horizontal gene transfer of stx genes in hybrid STEC/ETEC isolates, we identified the bacteriophage sequences using PHASTER. The results obtained for the 27 hybrid STEC/ETEC strains are summarized in Table 3. The presence of the majority of stx2 gene sequences was confirmed from phage sequence regions corresponding to “intact” (26/27). Additionally, it was confirmed that the sequences corresponding to the stx2 gene in one STEC/ETEC hybrid strain genome (MFDS1012367; score 90) were found in the “questionable” phage region.

3.6. Plasmid-Associated Sequence

To investigate the plasmid-mediated horizontal gene transfer of est in the hybrid STEC/ETEC isolates, we analyzed plasmid-associated sequences using PlasmidFinder 2.1. The plasmid replication results for the 27 hybrid STEC/ETEC isolates are summarized in Table 3. PlasmidFinder identified several plasmid replicon sequences of known Inc groups in all the STEC/ETEC genomes. We elucidated that each of the 27 genomes had an IncFIl or IncFIB plasmid origin and in some cases, such as MFDS1016200, both. The genes encoding heat-stable enterotoxin STa (estA) or STb (estB) were placed in the same contig as IncFIl and IncFIB. The mobile genetic elements proximal to estA and estB are shown in Supplementary Table S4.

3.7. Phylogenetic Analysis and Population Structure Analysis

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/ETEC hybrids and other pathogenic E. coli isolates. The 187 genome datasets included the sequencing results of 160 pathogenic E. coli as well as 27 hybrid STEC/ETEC genomes, which were deposited in the NCBI database. Phylogenetic tree analysis revealed that these hybrids were closely related to certain ETEC (21 strains, 77.8%) and STEC (six strains, 22.2%) strains, implying the potential acquisition of Stx-phages and/or ETEC virulence genes during their emergence (Figure 3A). In addition, the population structure of the 187 genome datasets was defined using the RhierBAPS, which divided the genome datasets into six primary sequence clusters (Bayesian analysis of population structure [BAPS] hierarchical level 1). These were further subdivided into 28 lineages (BAPS level 2) (Figure 3B). The results showed that of the total, 21 hybrid strains closely related to ETEC were divided into six groups (three level 1, five level 2), while the remaining six hybrids correlated with STEC were divided into two groups (two level 1, two level 2).

4. Discussion

STEC/ETEC hybrids have been recovered from various sources, including humans, animals, food, and water, some of which have been associated with diarrheal diseases and HUS in humans. In South Korea, the STEC/ETEC hybrid strain was first isolated from a patient suffering from diarrhea in 2014 [23]. Here, we report 27 STEC/ETEC hybrid strains among 1025 pathogenic E. coli strains identified in South Korea between 2016 and 2020. This study characterized the virulence and antibiotic resistance genes harbored by these hybrid strains, to further determine their phylogeny among other pathogenic E. coli strains. The molecular properties of these strains were investigated using real-time PCR followed by whole-genome sequencing (WGS). Phylogenetic analysis was performed to assess the phylogenetic positions of these hybrids in a diverse collection of pathogenic E. coli representing all the major pathotypes.
For the initial molecular characterization of all pathogenic E. coli strains, real-time PCR and serotyping were employed. Subsequent WGS analysis of these hybrids yielded results that were consistent with serotyping and the presence of virulence factors. The presence of genes encoding Shiga toxin 2 and heat-stable enterotoxin, namely, stx2 and est, respectively, was confirmed in all 27 STEC/ETEC hybrid strains. Most of the STEC/ETEC hybrid strains among human and animal isolates in Finland [25] harbored the stx2 gene without the stx1 gene. In addition, STEC/ETEC hybrid strains from diarrheal patients in South Korea [27] and Sweden [28] harbored the stx2 gene. However, the majority of STEC/ETEC hybrid strains in livestock of Bangladesh [26] carried the stx1 gene. Our results suggest that the STEC/ETEC hybrid strains isolated in South Korea that contained the stx2 gene may be more dangerous to humans. Shiga toxins are major factors contributing to the virulence of STEC; however, adhesion and colonization to the human intestine are required for STEC pathogenesis [42,43,44]. Some STEC strains carry the locus of enterocyte effacement (LEE-positive) [45,46,47], whereas those that do not carry the LEE (LEE-negative) and mainly harbor the locus of adhesion and autoaggregation (LAA) have also been associated with illness [48,49,50,51]. LAA is found either as a “complete” structure with four modules (module I (hes and other genes), module II (iha, lesP, and others genes), module III (pagC, tpsA, and other genes), and module IV (agn43 and other genes)) or as an “incomplete” structure if one of the modules is missing [48]. In this study, 27 STEC/ETEC hybrid strains carried one copy of the stx2 gene, lacked eae (E. coli attaching and effacing) gene and were LEE-negative STEC strains. Consistent with findings of previous studies, we observed some genes encoding LAA in some of the identified STEC/ETEC hybrid strains, except for seven strains (MFDS 1009773, 1012367, 1014122, 1016183, 1016224, 1016228, and 1016229).
In addition, the colonization of ETEC on the surface of the intestinal epithelium is a critical step in exerting its toxicity [52]. In addition to heat-labile (LT) and/or heat-stable (ST) enterotoxins, colonization factors (CFs) are major virulence factors in ETEC. Once ETEC colonizes the small intestinal epithelia through CFs, effective enterotoxin delivery commences, which is responsible for the secretion of water and electrolytes from the intestinal lumen [53,54]. These factors are referred to as colonization factor antigen I (CFA/I) or coli surface (CS) antigen [55]. The CFA/I of ETEC-related genes, such as cfa A, cfa B, cfa C, cfa D, and cfa E, was detected in 16 STEC/ETEC hybrid strains.
In addition, the hybrids represented diverse serotypes (O2:H25 (2), O2:H27 (1), O8:H8 (1), O8:H9 (4), O100:H30 (7), O141:H29 (1), O148:H7 (1), O155:H21 (4), O168:H8 (5), and O174:H2 (1)] and sequence types [ST446 (7), ST1021 (5), ST21 (4), ST74 (4), ST785 (1), ST670 (2), ST1780 (1), ST1782 (1), ST10 (1), and ST726 (1)). Previous studies have revealed the diversity of sequence types and serotypes (>40) among STEC/ETEC hybrid strains. This diversity suggests that both the ETEC virulence gene-carrying plasmids and Shiga toxin-containing bacteriophages could spread to a broad range of genetic backgrounds, including serotypes related to more pathogenic disease-causing strains such as O2:H27, O15:H16, O101:H-, O128:H8, and O141:H8 [25].
This study describes the virulence gene transfer of STEC/ETEC hybrid strains isolated from livestock feces and animal source foods in South Korea. It emphasizes that WGS is a powerful tool to analyze bacterial genomes for the presence of regions of MGEs, such as phages and plasmids, in them. Furthermore, the genomic information obtained in this study can significantly contribute to a better understanding of the genomic characteristics of hybrid E. coli strains in the future. In further studies, it may be necessary to investigate the genomic and transcriptome characteristics of STEC/ETEC hybrid strains isolated from diverse ecological and geographical sources in Korea.

5. Conclusions

In conclusion, we are the first to report the virulence and antibiotic resistance profiles of STEC/ETEC hybrid strains isolated from livestock feces (cattle and pigs) and animal source foods (beef, pork, and meat patties) in South Korea. Through genome-based characterization, we confirmed that virulence markers present in STEC/ETEC pathotypes were carried by MGEs, such as phages and plasmids. In addition, we identified adhesion and colonization factors in the human intestine required for STEC pathogenesis. Most DEC is subdivided into several pathotypes based on the presence of specific virulence traits directly related to disease development [3,4]. Importantly, our results emphasize that the hybrid strains of E. coli with STEC and other DEC-associated virulence factors may be more dangerous than STEC alone [14,20,21,22,23,24,25,26,27,28]. Thus, the emergence of hybrid DEC strains may have severe consequences for public health and should be considered in patient care and epidemiological surveillance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11051285/s1, Table S1: Virulence genes in hybrid STEC/ETEC strain genomes predicted by VFDB; Table S2: Antibiotic resistance genes in hybrid STEC/ETEC strain genomes predicted by ResFinder; Table S3: The phenotypic profile of antimicrobial resistance in hybrid STEC/ETEC strains; Table S4: Mobile genetic elements proximal to genes encoding STa (estA) or STb (estB); Table S5: Summarized characteristics of pathogenic E. coli strains.

Author Contributions

Conceptualization, S.H.K. (Soon Han Kim), H.-Y.K. and S.H.K. (Seung Hwan Kim); methodology, W.L., M.-H.K., S.S., E.K. and E.S.A.; data curation, W.L., M.-H.K. and S.S.; writing—original draft preparation, W.L.; writing—review and editing, E.K., S.H.K. (Soon Han Kim) and H.-Y.K.; supervision, S.H.K. (Soon Han Kim) and H.-Y.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Ministry of Food and Drug Safety (grant numbers 20161MFDS030 and 23194MFDS017). The findings and conclusions of this study are our own and do not necessarily represent the views of the Ministry of Food and Drug Safety.

Data Availability Statement

Sequence data have been submitted to the publicly accessible NCBI archives (https://ncbi.nlm.nih.gov (accessed on 25 January 2023), including GenBank and Sequence Read Archive (SRA), under the accession numbers listed in Table 1 and Supplementary Table S5.

Conflicts of Interest

The authors declare no conflict of interest.

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Microorganisms 11 01285 g001 550
Figure 1. The heatmap of virulence factors across the hybrid Shiga toxin-producing Escherichia coli (STEC)/enterotoxigenic E. coli (ETEC) strains. Heatmap showing the presence/absence of virulence factors (y-axis) among the hybrid STEC/ETEC isolates identified in this study (x-axis). The presence of virulence genes is shown in dark blue, whereas the absence is shown in light blue, as indicated in the color key. Heatmap was generated using the gplot (v3.1.3) package in R software (v4.1.3).
Figure 1. The heatmap of virulence factors across the hybrid Shiga toxin-producing Escherichia coli (STEC)/enterotoxigenic E. coli (ETEC) strains. Heatmap showing the presence/absence of virulence factors (y-axis) among the hybrid STEC/ETEC isolates identified in this study (x-axis). The presence of virulence genes is shown in dark blue, whereas the absence is shown in light blue, as indicated in the color key. Heatmap was generated using the gplot (v3.1.3) package in R software (v4.1.3).
Microorganisms 11 01285 g001
Microorganisms 11 01285 g002 550
Figure 2. The heatmap of antimicrobial profiles across the hybrid STEC/ETEC strains. Heatmap showing the presence/absence of antimicrobial resistance (y-axis) among the hybrid STEC/ETEC isolates identified in this study (x-axis). The presence of antimicrobial genes is shown in bright purple, whereas absence is shown in light purple, as indicated in the color key. Heatmap was generated using the gplot (v3.1.3) package in R software (v4.1.3).
Figure 2. The heatmap of antimicrobial profiles across the hybrid STEC/ETEC strains. Heatmap showing the presence/absence of antimicrobial resistance (y-axis) among the hybrid STEC/ETEC isolates identified in this study (x-axis). The presence of antimicrobial genes is shown in bright purple, whereas absence is shown in light purple, as indicated in the color key. Heatmap was generated using the gplot (v3.1.3) package in R software (v4.1.3).
Microorganisms 11 01285 g002
Microorganisms 11 01285 g003 550
Figure 3. Phylogenetic analysis and population structure analysis of hybrid STEC/ETEC strains. (A) Strains are colored based on the pathogenic E. coli groups: blue, STEC strains; pink, ETEC strains; gray, EPEC strains; green, EAEC strain; and brown, EIEC strains. The pink and blue bars represent 21 and 6 STEC/ETEC hybrid strains that are closely related to specific ETEC and STEC strains, respectively. (B) Sequence clusters (1 to 6) are indicated in the outer colored dot, which are further divided into 28 lineages (inner ring). Of the total, 21 hybrid strains closely related to ETEC were divided into six groups (three level 1, five level 2), while the remaining 6 hybrids correlated with STEC were divided into two groups (two level 1, two level 2).
Figure 3. Phylogenetic analysis and population structure analysis of hybrid STEC/ETEC strains. (A) Strains are colored based on the pathogenic E. coli groups: blue, STEC strains; pink, ETEC strains; gray, EPEC strains; green, EAEC strain; and brown, EIEC strains. The pink and blue bars represent 21 and 6 STEC/ETEC hybrid strains that are closely related to specific ETEC and STEC strains, respectively. (B) Sequence clusters (1 to 6) are indicated in the outer colored dot, which are further divided into 28 lineages (inner ring). Of the total, 21 hybrid strains closely related to ETEC were divided into six groups (three level 1, five level 2), while the remaining 6 hybrids correlated with STEC were divided into two groups (two level 1, two level 2).
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Table
Table 1. Summarized characteristics of hybrid Shiga toxin-producing Escherichia coli (STEC)/enterotoxigenic E. coli (ETEC) strains.
Table 1. Summarized characteristics of hybrid Shiga toxin-producing Escherichia coli (STEC)/enterotoxigenic E. coli (ETEC) strains.
Strain NameCollection DateGeographic
Location		Isolation SourceCoverageContigsSize (bp)GC (%)CDSsrRNAtRNAAccession No.
MFDS10077848 June 2016Jeollanam-doanimal source foods (meat patties)17855,327,823 50.65465 22107JAQJCX000000000
MFDS100973631 August 2017Incheonlivestock feces24355,431,463 50.45439 2293JAQMUJ000000000
MFDS100976020 May 2017Incheonlivestock feces
(pig)		28885,332,414 50.65411 2291JAQMUK000000000
MFDS10097644 July 2017Incheonlivestock feces
(pig)		126145,355,898 50.55467 2289JAQMUL000000000
MFDS100977018 July 2017Incheonlivestock feces
(pig)		18245,322,048 50.65419 2291JAQMUM000000000
MFDS100977325 July 2017Incheonlivestock feces
(cattle)		17675,310,620 50.65419 22100JAQMUO000000000
MFDS101099231 January 2018Jeollabuk-doanimal source foods (beef)25165,808,504 50.45998 22100JAQMUP000000000
MFDS101236728 December 2018Jeju-doanimal source foods (beef)17755,253,992 50.75518 2294JAQMUR000000000
MFDS101378112 February 2019Chungcheongnam-dolivestock feces
(pig)		27875,392,379 50.65517 2289JAQMUS000000000
MFDS101378212 February 2019Incheonlivestock feces
(pig)		14285,374,310 50.45550 2294JAQMUT000000000
MFDS10138369 April 2019Chungcheongnam-dolivestock feces
(pig)		27565,685,673 50.55819 2292JAQMUU000000000
MFDS101386431 May 2019Chungcheongnam-dolivestock feces
(pig)		181155,865,149 50.56141 2294JAQMUV000000000
MFDS101392115 November 2019Jeollabuk-doanimal source foods (beef)231115,498,083 50.65669 22100JAQMUW000000000
MFDS10141227 January 2019Gwangjuanimal source foods (beef)17755,462,265 50.35487 2298JAQMUX000000000
MFDS10141829 July 2019Gyeongsangbuk-doanimal source foods (beef)235115,557,991 50.55767 22101JAQMUY000000000
MFDS101593910 February 2020Gwangjuanimal source foods (beef)31955,406,905 50.55520 2296JAQMUZ000000000
MFDS101618324 March 2020Gyeonggi-dolivestock feces
(pig)		43845,064,469 50.95087 2292JAQMVA000000000
MFDS101619527 March 2020Daejeonlivestock feces
(cattle)		24695,789,890 50.56119 2289JAQMVB000000000
MFDS101620027 March 2020Daejeonlivestock feces
(cattle)		524155,393,111 50.95542 2297JAQMVC000000000
MFDS10162025 May 2020Chungcheongnam-dolivestock feces
(pig)		516105,451,302 50.65662 2298JAQMVD000000000
MFDS101622214 April 2020Chungcheongnam-dolivestock feces
(pig)		19335,088,334 50.85131 2292JAQMVE000000000
MFDS101622414 April 2020Chungcheongnam-dolivestock feces
(pig)		19855,304,302 50.85334 2288JAQMVF000000000
MFDS101622812 May 2020Gyeonggi-dolivestock feces
(pig)		38735,064,746 50.95087 2292JAQMVG000000000
MFDS101622912 May 2020Gyeonggi-dolivestock feces
(pig)		19735,068,005 50.95081 2291JAQMVH000000000
MFDS10162335 May 2020Chungcheongnam-dolivestock feces
(pig)		36465,741,335 50.45889 2292JAQMVI000000000
MFDS101623512 May 2020Gyeonggi-dolivestock feces
(pig)		15485,232,588 50.85295 2291JAQMVJ000000000
MFDS101641623 Mar 2020Jeollabuk-doanimal source foods (pork)29075,138,577 50.75183 2290JAQMVK000000000
Table
Table 2. Serotypes and sequence types of the hybrid STEC/ETEC strains.
Table 2. Serotypes and sequence types of the hybrid STEC/ETEC strains.
Strain NameCollection DateSerotypeSequence Type
MFDS10077848 June 2016O2H27ST10
MFDS100973631 August 2017O148H7ST1780
MFDS100976020 May 2017O100H30ST446
MFDS10097644 July 2017O100H30ST446
MFDS100977018 July 2017O100H30ST446
MFDS100977325 July 2017O168H8ST1021
MFDS101099231 January 2018O168H8ST1021
MFDS101236728 December 2018O2H25ST670
MFDS101378112 February 2019O100H30ST446
MFDS101378212 February 2019O8H8ST74
MFDS10138369 April 2019O8H9ST1782
MFDS101386431 May 2019O8H9ST74
MFDS101392115 November 2019O168H8ST1021
MFDS10141227 January 2019O2H25ST670
MFDS10141829 July 2019O168H8ST1021
MFDS101593910 February 2020O168H8ST1021
MFDS101618324 March 2020O155H21ST21
MFDS101619527 March 2020O8H9ST74
MFDS101620027 March 2020O141H29ST785
MFDS10162025 May 2020O155H21ST21
MFDS101622214 April 2020O100H30ST446
MFDS101622414 April 2020O174H2ST726
MFDS101622812 May 2020O155H21ST21
MFDS101622912 May 2020O155H21ST21
MFDS10162335 May 2020O8H9ST74
MFDS101623512 May 2020O100H30ST446
MFDS101641623 March 2020O100H30ST446
Table
Table 3. Phage and plasmid replicons of hybrid STEC/ETEC strains.
Table 3. Phage and plasmid replicons of hybrid STEC/ETEC strains.
Strain NamePhagePlasmid Replicon
stx SubtypeCompletenessScore
MFDS1007784stx2A, stx2Bintact150IncFIB
MFDS1009736stx2A, stx2Bintact150IncFIB
MFDS1009760stx2A, stx2Bintact150IncFIl
MFDS1009764stx2A, stx2Bintact150IncFIl
MFDS1009770stx2A, stx2Bintact150IncFIl
MFDS1009773stx2A, stx2Bintact110IncFIB
MFDS1010992stx2A, stx2Bintact150IncFIB
MFDS1012367stx2A, stx2Bquestionable90IncFIB
MFDS1013781stx2A, stx2Bintact150IncFIl
MFDS1013782stx2A, stx2Bintact150IncFIl
MFDS1013836stx2A, stx2Bintact150IncFIl
MFDS1013864stx2A, stx2Bintact150IncFIl
MFDS1013921stx2A, stx2Bintact150IncFIB
MFDS1014122stx2A, stx2Bintact110IncFIB
MFDS1014182stx2A, stx2Bintact130IncFIB
MFDS1015939stx2A, stx2Bintact150IncFIB
MFDS1016183stx2A, stx2Bintact150IncFIl
MFDS1016195stx2A, stx2Bintact150IncFIl, IncR, IncX1
MFDS1016200stx2A, stx2Bintact100IncFIl, IncX1, IncFIB
MFDS1016202stx2A, stx2Bintact150IncFIl
MFDS1016222stx2A, stx2Bintact140IncFIl
MFDS1016224stx2A, stx2Bintact150IncFIl
MFDS1016228stx2A, stx2Bintact150IncFIl
MFDS1016229stx2A, stx2Bintact150IncFIl
MFDS1016233stx2A, stx2Bintact150IncFIl
MFDS1016235stx2A, stx2Bintact140IncFIl
MFDS1016416stx2A, stx2Bintact150IncFIl
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Lee, W.; Kim, M.-H.; Sung, S.; Kim, E.; An, E.S.; Kim, S.H.; Kim, S.H.; Kim, H.-Y. Genome-Based Characterization of Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains Isolated in South Korea, 2016–2020. Microorganisms 2023, 11, 1285. https://doi.org/10.3390/microorganisms11051285

AMA Style

Lee W, Kim M-H, Sung S, Kim E, An ES, Kim SH, Kim SH, Kim H-Y. Genome-Based Characterization of Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains Isolated in South Korea, 2016–2020. Microorganisms. 2023; 11(5):1285. https://doi.org/10.3390/microorganisms11051285

Chicago/Turabian Style

Lee, Woojung, Min-Hee Kim, Soohyun Sung, Eiseul Kim, Eun Sook An, Seung Hwan Kim, Soon Han Kim, and Hae-Yeong Kim. 2023. "Genome-Based Characterization of Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains Isolated in South Korea, 2016–2020" Microorganisms 11, no. 5: 1285. https://doi.org/10.3390/microorganisms11051285

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