Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea.

The emergence of transferable linezolid resistance genes poses significant challenges to public health, as it does not only confer linezolid resistance but also reduces susceptibility to florfenicol, which is widely used in the veterinary field. This study evaluated the genetic characteristics of linezolid-resistant Staphylococcus aureus strains isolated from pig carcasses and further clarified potential resistance and virulence mechanisms in a newly identified sequence type. Of more than 2500 strains isolated in a prior study, 15 isolated from pig carcasses exhibited linezolid resistance (minimum inhibitory concentration ≥ 8 mg/L). The strains were characterized in detail by genomic analysis. Linezolid-resistant S. aureus strains exhibited a high degree of genetic lineage diversity, with one strain (LNZ_R_SAU_64) belonging to ST8004, which has not been reported previously. The 15 strains carried a total of 21 antibiotic resistance genes, and five carried mecA associated with methicillin resistance. All strains harbored cfr and fexA, which mediate resistance to linezolid, phenicol, and other antibiotics. Moreover, the strains carried enterotoxin gene clusters, including the hemolysin, leukotoxin, and protease genes, which are associated with humans or livestock. Some genes were predicted to be carried in plasmids or flanked by ISSau9 and the transposon Tn554, thus being transmittable between staphylococci. Strains carrying the plasmid replicon repUS5 displayed high sequence similarity (99%) to the previously reported strain pSA737 in human clinical samples in the United States. The results illustrate the need for continuous monitoring of the prevalence and transmission of linezolid-resistant S. aureus isolated from animals and their products.

Weissella and the two Janus faces of the genus.

The genus Weissella belongs to the lactic acid bacteria group. It occurs naturally in foods and is a component of the human microbiome. A few Weissella species are candidate probiotics due to their potential for survival under the harsh conditions present in the gastrointestinal tract of humans and animals. Various species have also shown potential for treating and preventing periodontal disease, skin pathologies, and atopic dermatitis; some are used as starters for the fermentation of foods due to their production of exopolysaccharides; and others are used as protective cultures due to their production of weissellicin, a bacteriocin. However, a few Weissella species are opportunistic pathogens, such as W. ceti, which is the etiological agent of weissellosis, a disease in rainbow trout. Additionally, most Weissella species are intrinsically vancomycin-resistant. Thus, the Weissella genus is important from both medical and industrial points of view, and the Janus faces of this genus should be considered in any expected biotechnological applications. In this review, we present an overview of the probiotic potential and pathogenic cases of the Weissella genus reported in the literature.

Comprehensive metagenomic analysis of stress-resistant and -sensitive Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium which can live in adverse environments (low pH, high salinity, and low temperature). Even though there are various whole genome sequencing (WGS) data on L. monocytogenes, investigations on genetic differences between stress-resistant and -sensitive L. monocytogenes grown under stress environments have been not fully examined. This study aims to investigate and compare genetic characteristics between stress-resistant and -sensitive L. monocytogenes using whole genome sequencing (WGS). A total of 47 L. monocytogenes strains (43 stress-resistant and 4 stress-sensitive) were selected based on the stress-resistance tests under pH 3, 5% salt concentration, and 1 °C. The sequencing library for WGS was prepared and sequenced using an Illumina MiSeq. Genetic characteristics of two different L. monocytogenes groups were examined to analyze the pangenome, functionality, virulence, antibiotic resistance, core, and unique genes. The functionality of unique genes in the stress-resistant L. monocytogenes was distinct compared to the stress-sensitive L. monocytogenes, such as carbohydrate and nucleotide transport and metabolism. The lisR virulence gene was detected more in the stress-resistant L. monocytogenes than in the stress-sensitive group. Five stress-resistant L. monocytogenes strains possessed tet(M) antibiotic resistance gene. This is the first study suggesting that deep genomic characteristics of L. monocytogenes may have different resistance level under stress conditions. This new insight will aid in understanding the genetic relationship between stress-resistant and -sensitive L. monocytogenes strains isolated from diverse resources. KEY POINTS: • Whole genomes of L. monocytogenes isolated from three different sources were analyzed. • Differences in two L. monocytogenes groups were identified in functionality, virulence, and antibiotic resistance genes. • This study first examines the association between resistances and whole genomes of stress-resistant and -sensitive L. monocytogenes.

Droplet digital PCR method for the absolute quantitative detection and monitoring of Lacticaseibacillus casei.

Droplet digital polymerase chain reaction (ddPCR) is an emerging molecular detection assay that provides an absolute quantification of targets. Despite its emerging applications in the detection of food microorganisms, there are limited reports of its use for the monitoring of microorganisms utilized as starters in the dairy industry. This study investigated the applicability of ddPCR as a detection platform for Lacticaseibacillus casei, a probiotic found in fermented foods and exerts beneficial effects on human health. In addition, this study compared the performance of ddPCR with that of real-time PCR. The ddPCR targeting the haloacid dehalogenase-like hydrolase (LBCZ_1793) exhibited high specificity against 102 nontarget bacteria, including Lacticaseibacillus species that is very closely related to L. casei. The ddPCR exhibited high linearity and efficiency within the quantitation range (105-100 CFU/ml), with the limit of detection being 100 CFU/ml. The ddPCR also demonstrated a higher sensitivity than real-time PCR in detecting low bacterial concentration in spiked milk samples. Furthermore, it provided an accurate absolute quantification of the concentration of L. casei, without the need for standard calibration curves. This study demonstrated that ddPCR is a useful method for monitoring starter cultures in dairy fermentations and detecting L. casei in foods.

Differentiation between Weissella cibaria and Weissella confusa Using Machine-Learning-Combined MALDI-TOF MS.

Although Weissella cibaria and W. confusa are essential food-fermenting bacteria, they are also opportunistic pathogens. Despite these species being commercially crucial, their taxonomy is still based on inaccurate identification methods. In this study, we present a novel approach for identifying two important Weissella species, W. cibaria and W. confusa, by combining matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer (MALDI-TOF MS) data using machine-learning techniques. After on- and off-plate protein extraction, we observed that the BioTyper database misidentified or could not differentiate Weissella species. Although Weissella species exhibited very similar protein profiles, these species can be differentiated on the basis of the results of a statistical analysis. To classify W. cibaria, W. confusa, and non-target Weissella species, machine learning was used for 167 spectra, which led to the listing of potential species-specific mass-to-charge (m/z) loci. Machine-learning techniques including artificial neural networks, principal component analysis combined with the K-nearest neighbor, support vector machine (SVM), and random forest were used. The model that applied the Radial Basis Function kernel algorithm in SVM achieved classification accuracy of 1.0 for training and test sets. The combination of MALDI-TOF MS and machine learning can efficiently classify closely-related species, enabling accurate microbial identification.

Molecular and Genomic Analysis of the Virulence Factors and Potential Transmission of Hybrid Enteropathogenic and Enterotoxigenic Escherichia coli (EPEC/ETEC) Strains Isolated in South Korea.

Hybrid strains Escherichia coli acquires genetic characteristics from multiple pathotypes and is speculated to be more virulent; however, understanding their pathogenicity is elusive. Here, we performed genome-based characterization of the hybrid of enteropathogenic (EPEC) and enterotoxigenic E. coli (ETEC), the strains that cause diarrhea and mortality in children. The virulence genes in the strains isolated from different sources in the South Korea were identified, and their phylogenetic positions were analyzed. The EPEC/ETEC hybrid strains harbored eae and est encoding E. coli attaching and effacing lesions and heat-stable enterotoxins of EPEC and ETEC, respectively. Genome-wide phylogeny revealed that all hybrids (n = 6) were closely related to EPEC strains, implying the potential acquisition of ETEC virulence genes during ETEC/EPEC hybrid emergence. The hybrids represented diverse serotypes (O153:H19 (n = 3), O49:H10 (n = 2), and O71:H19 (n = 1)) and sequence types (ST546, n = 4; ST785, n = 2). Furthermore, heat-stable toxin-encoding plasmids possessing estA and various other virulence genes and transporters, including nleH2, hlyA, hlyB, hlyC, hlyD, espC, espP, phage endopeptidase Rz, and phage holin, were identified. These findings provide insights into understanding the pathogenicity of EPEC/ETEC hybrid strains and may aid in comparative studies, virulence characterization, and understanding evolutionary biology.

Identification of novel molecular targets for Weissella species-specific real-time PCR based on pangenome analysis.

Some Weissella species are used in probiotic products because of their beneficial effects in humans, whereas some species are considered as opportunistic pathogens that cause infections in humans. Therefore, an accurate and rapid identification of Weissella species is essential to control pathogenic Weissella species or isolate new functional strains with probiotic effects from their habitat. The objective of our study was to extract novel molecular targets using pangenome analysis for the identification of major Weissella species present in food. With 50 genomes representing 11 Weissella species, novel molecular targets were mined based on their 100% presence in the respective strains of the target species and absence in the strains of non-target bacteria. Primers based on molecular targets showed positive results for the corresponding species, whereas 79 non-target strains showed negative results. Standard curves revealed good linearity in the range of 103-108 colony-forming units per reaction. Our method was successfully applied to 74 Weissella strains isolated from food samples to demonstrate that the molecular targets provided a viable alternative to the 16S rRNA sequence. Furthermore, it was possible to identify and quantify Weissella communities in fermented foods. These results demonstrate that our method can be used for effective and accurate screening for the presence of Weissella species in foods. KEY POINTS: • This is first study to mine novel targets for differentiating 11 Weissella species. • The novel targets showed higher resolution than the 16S rRNA gene sequence. • The PCR method effectively detected Weissella species with opposing properties.

Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the discrimination of Lacticaseibacillus species.

The closely related species, Lacticaseibacillus casei, L. paracasei, L. rhamnosus, L. chiayiensis, and L. zeae, are difficult to accurately discriminate by conventional identification methods. In this study, the bioTyper and in-house database of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated to discriminate five Lacticaseibacillus species. From the mass spectra of 130 isolates aligned with databases, 118 strains were correctly identified. On the other hand, databases could not accurately differentiate 12 isolates such as L. casei, L. rhamnosus and L. chiayiensis because the same colony was identified as two species with similar score. To overcome the database's limitations, the mass spectra were analyzed to discover species-specific protein peaks. The peaks at 6731 ± 1, 6849 ± 1, 7008 ± 1, 7376 ± 1, and 2593 ± 1 m/z were specifically found in the reference strains of L. casei, L. paracasei, L. rhamnosus, L. chiayiensis, and L. zeae, respectively. These peaks confirmed that the five peaks were consistently present in each species using 130 strains isolated from food samples. Our results demonstrate the high-resolution of MALDI-TOF MS technique for rapid and accurate classification of five species when used with databases coupled to specific peaks.

Novel approaches for the identification of microbial communities in kimchi: MALDI-TOF MS analysis and high-throughput sequencing.

Complex interactions occur within microbial communities during the fermentation process of kimchi. Identification of these microorganisms provides the essential information required to improve food quality and to understand their role in this process. This was the first study to compare two methods for accuracy in the identification of microbial community changes during the fermentation of kimchi by comparing a culture-dependent (MALDI-TOF MS analysis) and a culture-independent method (high-throughput sequencing) of 16S rRNA gene fragment). Members of the Lactobacillus-related genera, Leuconostoc, and Weissella were identified as the predominant microorganisms by both methods. The culture-independent method was able to additionally identify non-lactic acid bacteria and yeasts, such as Kazachstania in kimchi. However, high-throughput sequencing failed to accurately recognize Latilactobacillus sakei, Latilactobacillus curvatus, Lactiplantibacillus plantarum, and W. cibaria, which played an important role in kimchi fermentation, as this method only allowed for identification at the genus level. Conversely, MALDI-TOF MS analysis could identify the isolates at the species level. Also, culture-dependent method could identify predominant species in viable cell communities. The culture-dependent method and culture-independent method provided complementary information by producing a more comprehensive view of the microbial ecology in fermented kimchi.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate.

BACKGROUND: Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. RESULTS: To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S-23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S-23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. CONCLUSIONS: The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

Complete Genome Sequence of Lactobacillus plantarum EM, A Putative Probiotic Strain with the Cholesterol-Lowering Effect and Antimicrobial Activity.

Lactobacillus plantarum EM is a probiotic strain with antimicrobial activity, cholesterol-lowering effects, and tolerance to acid and bile. To understand the genetic basis of the probiotic characteristics of this strain, genome sequencing and probiotic-related genetic analysis were performed. The genomic characteristics of L. plantarum EM were confirmed by comparative genomic analysis with 41 probiotic lactic acid bacteria, including 10 L. plantarum strains. L. plantarum EM was shown to contain a circular chromosome of 3,184,808 bp and eight plasmids with various lengths from 5,027 to 76,369 bp. The L. plantarum EM genome had a total of 3560 protein-coding genes, including probiotic-related genes, such as tolerance to acid and bile, temperature stress, and oxidative stress. Comparative genomic analysis showed that L. plantarum EM contained plantaricin and bovicin gene clusters, which are related to antimicrobial activity, and five bile salt hydrolase genes related to serum cholesterol-lowering effects. The genomic analysis confirmed the probiotic properties of L. plantarum EM, and our results indicated that this strain has potential application for use as an industrially important probiotic.

Novel real-time PCR assay for Lactobacillus casei group species using comparative genomics.

The Lactobacillus casei group, which includes the closely related species L. casei, L. paracasei, L. rhamnosus, and L. chiayiensis, has been under debate regarding its taxonomy because of the difficulty in distinguishing the species from each other. In the present study, we developed a novel real-time PCR assay for distinguishing the L. casei group species. The pan-genome, as determined by the genomes of 44 strains, comprised 6789 genes, comparative genomic analysis showed that L. casei group strains were classified by species. Based on these results, species-specific genes were identified, and primers were designed from those genes. Real-time PCR clearly distinguished each species of the L. casei group and specifically amplified only to the target species. The method was applied to 29 probiotic products, and the detected results and label claims were compared. Total 23 products were in accordance with the label claims, and the remaining products contained species different from those stated in the label claims. Our method can rapidly and accurately distinguish the L. casei group species in a single reaction. Hence, our assay can be applied to identify L. casei group species from food or environmental samples and to accurately determine the nomenclature of the species.

Lactobacillus terrae sp. nov., a novel species isolated from soil samples in the Republic of Korea.

A novel strain, designated NIBRBAC000499792T, was isolated from a soil sample collected at Jukgye, Dongnam, Cheonan, Republic of Korea. Cells were Gram-positive, non-motile, non-spore-forming, rod-shaped, oxidase-negative and catalase-negative. Colonies grown on de Man, Rogosa and Sharpe agar were white, circular, raised and entire. Analysis of the 16S rRNA gene sequence analysis revealed that strain NIBRBAC000499792T belongs to the genus Lactobacillus (family Lactobacillaceae) and is most closely related to Lactobacillus nodensis DSM 19682T (96.1 % similarity) and Lactobacillus tucceti KCTC 21005T (96.7 %). The results of DNA-DNA hybridization experiments demonstrated that strain NIBRBAC000499792T represents a novel species. Major fatty acids are C18 : 1ω9c, C16 : 0 and unidentified 18.846 and/or C19 : 1ω6c and/or C19 : 0cyclo. The predominant respiratory quinones are menaquinone-8 and menaquinone-9. The major polar lipids are phosphatidylglycerol and diphosphatidylglycerol. The minor polar lipids are one unidentified aminophospholipid, one unidentified phospholipid, and four unidentified lipids. Next-generation sequencing analysis of strain NIBRBAC000499792T indicated that the total genome size was 1 548 794 bp with a G+C content of 33.1 mol%, 1586 coding sequences, 50 tRNAs and nine rRNAs. The most closely related genomes belonged to Lactobacillus species. Most metabolic pathways were related to carbon metabolism and carbon fixation. Based on this polyphasic analysis, strain NIBRBAC000499792T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus terrae sp. nov. is proposed, with the type strain NIBRBAC000499792T (=KCTC 21093T=JCM 32269T).

Rapid and accurate identification of species of the genus Pediococcus isolated from Korean fermented foods by matrix-assisted laser desorption/ionization time-of-flight MS with local database extension.

Pediococci are halophilic lactic acid bacteria, within the family Lactobacillaceae, which are involved in the fermentation of various salted and fermented foods, such as kimchi and jeotgal. In this study, a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS method was developed for the rapid identification of species of the genus Pediococcus. Of the 130 Pediococcus spectra aligned with the Biotyper taxonomy database, 122 isolates (93.9 %) yielded log scores <1.7, which means they were not identifiable. After registering the spectra of 11 reference strains of the genus Pediococcus, all of the isolates were correctly identified, of which 84 (64.6 %) and 46 (35.4 %) were identified at the species and genus level, respectively. In comparing food origins, no relationship was found between the bacterial characteristics and food environment. We were able to produce a Biotyper system for identification of members of the genus Pediococcus with locally extended Pediococcus reference strains. The MALDI-TOF MS method is fast, simple and reliable for discriminating between species in the genus Pediococcus and therefore will be useful for quality control in determining the spoilage of alcoholic beverages or in the production of fermented food.

Multiplex PCR for detection of the Vibrio genus and five pathogenic Vibrio species with primer sets designed using comparative genomics.

BACKGROUND: The genus Vibrio is clinically significant and major pathogenic Vibrio species causing human Vibrio infections are V. cholerae, V. parahaemolyticus, V. vulnificus, V. alginolyticus and V. mimicus. In this study, we screened for novel genetic markers using comparative genomics and developed a Vibrio multiplex PCR for the reliable diagnosis of the Vibrio genus and the associated major pathogenic Vibrio species. METHODS: A total of 30 Vibrio genome sequences were subjected to comparative genomics, and specific genes of the Vibrio genus and five major pathogenic Vibrio species were screened. The designed primer sets from the screened genes were evaluated by single PCR using DNAs from various Vibrio spp. and other non-Vibrio bacterial strains. A sextuplet multiplex PCR using six primer sets was developed to enable detection of the Vibrio genus and five pathogenic Vibrio species. RESULTS: The designed primer sets from the screened genes yielded specific diagnostic results for target the Vibrio genus and Vibrio species. The specificity of the developed multiplex PCR was confirmed with various Vibrio and non-Vibrio strains. This Vibrio multiplex PCR was evaluated using 117 Vibrio strains isolated from the south seashore areas in Korea and Vibrio isolates were identified as Vibrio spp., V. parahaemolyticus, V. vulnificus and V. alginolyticus, demonstrating the specificity and discriminative ability of the assay towards Vibrio species. CONCLUSIONS: This novel multiplex PCR method could provide reliable and informative identification of the Vibrio genus and major pathogenic Vibrio species in the food safety industry and in early clinical treatment, thereby protecting humans against Vibrio infection.

Rapid and Simultaneous Authentication of Six Laver Species Using Capillary Electrophoresis-Based Multiplex PCR.

Lavers are typically consumed in dried or seasoned forms. However, commercially processed lavers can lead to seafood fraud because it is impossible to authenticate the original species based on morphological characteristics alone. In this study, we developed a capillary electrophoresis-based multiplex polymerase chain reaction (PCR) to authenticate six different laver species. The species-specific primer sets to target the chloroplast rbcL or rbcS genes were newly designed. We successfully established both singleplex and multiplex conditions, which resulted in specific amplicons for each species (N. dentata, 274 bp; N. yezoensis, 211 bp; N. seriata, 195 bp; N. tenera, 169 bp; N. haitanensis, 127 bp; P. suborbiculata, 117 bp). Moreover, the assays were sensitive enough to detect DNA ranging from 10 to 0.1 pg of DNA. The optimized capillary electrophoresis-based multiplex PCR was successfully applied to 40 commercial laver products. In addition to detecting the laver species as stated on the commercial label, the assay discovered cases where less expensive species were mixed in. With its advantageous properties, such as short amplicon size, high specificity, and superior sensitivity, this assay could be used for the authentication of the six laver species.

Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

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.