Enteropathogenic Escherichia coli (EPEC) are a major bacterial cause of diarrhea worldwide. EPEC infection causes acute watery diarrhea accompanied by dehydration, vomiting, and fever (Kaper et al., 2004). The primary mechanism of EPEC infection is attachment to epithelial cells via bundle-forming pili (bfp) and tight attachment with the help of translocated intimin receptor (tir) and intimin (eae), resulting in accumulation of actin and formation of pedestal structures (Ledwaba et al., 2020). This study presents the complete genome sequence of an EPEC strain collected from Osan, South Korea, in 2021.

EPEC MFDS1019811 was identified and obtained during food poisoning investigation conducted by the Ministry of Food and Drug Safety (MFDS, South Korea) following a reported outbreak at a high school in Osan in 2021. The strain was isolated from donut and incubated on tryptic soy agar medium at 37℃ for 24 h. Genomic DNA was isolated using the Bioneer Genomic DNA Extraction Kit. Using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Fisher Scientific) and a Qubit 3.0 Fluorometer (Invitrogen), the extracted DNA was quantified. Complete genome sequencing was performed using two sequencing facilities: Illumina MiSeq and Oxford Nanopore MinION. The Nextera DNA Flex Library Prep Kit (Illumina) was used for library preparation on Miseq. The size of the prepared library was confirmed using a Bioanalyzer 2100 (Agilent Technologies). The paired-end library sequencing was performed using the MiSeq system with MiSeq Reagent Kit v3 (600-cycles). Individual sequence reads were analyzed using FastQC-v.0.11.8 post-sequencing. For Nanopore sequencing, a library was constructed using a Native Barcoding Kit24 V14 (Oxford Nanopore Technologies) and basecalling performed on guppy_barcoder v6.0.1. Illumina and nanopore sequencing data were

processed and hybrid-assembled using Unicycler v0.4.9. The assembled genome was then annotated using the RASTtk, through the BV-BRC web service (v3.32.13a). The Virulence Factor Database (VFDB) (Liu et al., 2022) and PATRIC_VF (Snyder et al., 2007) were used to predict the virulence-associated genes. MFDS1019811 genome comprises of 5,077,766 bp (50.67% GC contents) chromosome and 50,011 bp (50.02% GC contents) plasmid. 5,048 and 90 genes were identified, respectively (Table 1 and Fig. 1).

Table 1

Genome features of EPEC MFDS1019811

Genomic features	Chromosome	Plasmid
Contig	2	1
Genome size (bp)	5,077,766	50,011
GC content (%)	50.67	50.02
Total number of CDSs	5,048	90
tRNA genes	92	0
rRNA genes	22	0

Fig. 1. Genome map of enteropathogenic Escherichia coli MFDS1019811 chromosomal DNA and plasmid. The graphical circular maps are shown of the alignment of genes and other genomic data. The tracks on the viewer are displayed as concentric rings, from outermost to innermost: CDS, AMR genes, VF genes, GC content, GC skew. Genes with specialized functions are labeled with different colors. Virulence-related genes in yellow, and antibiotic resistance in red.

Formation of attaching and effacing (A/E) lesion in gut epithelium necessitates the presence of enterocyte effacement (LEE) in the bacterial genome. This locus encodes intimin (eae), a type III secretion system (T3SS) and six effectors, including the indispensable translocated intimin receptor (Tir) (Kaper et al., 2004). Additionally, typical EPEC possess E. coli adherence factor (EAF) associated the bfp gene, whereas atypical EPEC lack this factor (Schmidt, 2010). MFDS1019811 harbored eae, tir and bfpA. These results suggested that this strain is typical EPEC. Moreover, MFDS1019811 contains several virulence-associated genes related to biofilm formation (agaBC, csgB, gatBCZ, kbaY), capsule biosynthesis (etk, etp, gfcABCDE), invasion (aslA, dam, ibeB, map, ompA, relA, yijP), and typical III secretion system (cesD, escCDFJNRSTUV, espB, sepDLQ) (Table 2). This information will prove valuable for investigating food-borne pathogens and offers a genetic foundation for a more detailed analysis of virulence factors.

Table 2

Virulence associated genes of EPEC MFDS1019811

Classification	Gene(s)	Predicted function	References
Biofilmformation	agaBC	PTS system, galactosamine-specific II component	Berlyn et al. (1998)
	csgB	Minor curlin subunit CsgB, nucleation component of curlin monomers	Berlyn et al. (1998)
	gatBCZ	PTS system, galactitol-specific II component (EC 2.7.1.200)	Berlyn et al. (1998)
	kbaY	D-tagatose-1,6-bisphosphate aldolase subunit KbaY (EC 4.1.2.40)	Berlyn et al. (1998)
Capsule biosynthesis	etk	Tyrosine-protein kinase (EC 2.7.10.2)	Ilan et al. (1999)
	etp	Low molecular weight protein-tyrosine-phosphatase (EC 3.1.3.48) => Etp	Vincent et al. (2000)
	gfcABCDE	Threonine-rich inner membrane protein Gfc	Larson et al. (2021)
Invasion	aslA	Arylsulfatase (EC 3.1.6.1)	Berlyn et al. (1998)
	dam	Methyl-directed repair DNA adenine methylase (EC 2.1.1.72)	Berlyn et al. (1998)
	ibeB	Copper/silver efflux RND transporter, outer membrane protein CusC	Berlyn et al. (1998)
	map	Putative chaperone (IpgB2)	Berlyn et al. (1998)
	ompA	Outer membrane protein A precursor	Berlyn et al. (1998)
	relA	Inactive (p)ppGpp 3'-pyrophosphohydrolase domain / GTP pyrophosphokinase (EC 2.7.6.5), (p)ppGpp synthetase I	Berlyn et al. (1998)
	tir	Hypothetical protein	Berlyn et al. (1998)
	yijP	Phosphoethanolamine transferase EptC [E. coli], specific for LPS heptose I residue	Berlyn et al. (1998)
Type III secretion system (TTSS)	cesD	Type III secretion chaperone protein for YopD (SycD)	Parkbin et al. (2021)
	escC	Type III secretion outermembrane pore forming protein (YscC, MxiD, HrcC, InvG)	Parkbin et al. (2021)
	escD	Type III secretion protein SsaD	Parkbin et al. (2021)
	escF	Type III secretion protein SsaG	Parkbin et al. (2021)
	escJ	Type III secretion bridge between inner and outermembrane lipoprotein (YscJ, HrcJ, EscJ, PscJ)	Parkbin et al. (2021)
	escN	Type III secretion cytoplasmic ATP synthase (EC 3.6.3.14, YscN, SpaL, MxiB, HrcN, EscN)	Parkbin et al. (2021)
	escR	Type III secretion inner membrane protein (YscR, SpaR, HrcR, EscR, homologous to flagellar export components)	Parkbin et al. (2021)
	escS	Type III secretion inner membrane protein (YscS, homologous to flagellar export components)	Parkbin et al. (2021)
	escT	Type III secretion inner membrane protein (YscT, HrcT, SpaR, EscT, EpaR1, homologous to flagellar export components)	Parkbin et al. (2021)
	escU	Type III secretion inner membrane protein (YscU, SpaS, EscU, HrcU, SsaU, homologous to flagellar export components)	Parkbin et al. (2021)
	escV	Type III secretion inner membrane channel protein (LcrD, HrcV, EscV, SsaV)	Parkbin et al. (2021)
	espB	Secretion system effector SseD	Parkbin et al. (2021)
	sepD	Type III secretion system protein SepD	Parkbin et al. (2021)
	sepL	Type III secretion cytoplasmic protein (YscL)	Parkbin et al. (2021)

Nucleotide sequence accession number(s)

Accession numbers for nucleotide sequence. The complete genome sequence of Escherichia coli MFDS1019811 has been deposited at the NCBI GenBank database under accession numbers JAWWMY000000000, and the strain has been deposited in the Korean Culture Collection for foodborne Pathogens under strain number MFDS1019811.

대장균은 장관감염증과 같은 식중독을 유발하는 식품매개 병원균 중 하나이다. 본 연구에서는 2021년 오산의 한 고등학교 급식에서 일어난 식중독 사고의 원인 식품으로 추정되는 도넛으로부터 분리된 Escherichia coli (MFDS1019811)의 유전체 분석을 수행하였다. Escherichia coli MFDS1019811은 5,077,766 bp 길이의 chromosomal DNA와 50,011 bp 길이의 plasmid로 구성되었으며, 각각의 G + C contents는 50.67%와 50.02%로 확인되었다. 유전자 예측 결과, 92개 tRNA, 22개 rRNA, 그리고 chromosome과 plasmid 각각 5,048개와 90개의 단백질 코딩유전자가 동정되었다. 또한, bundle-forming pili (bfp)와 intimin (eae)를 포함한 여러 병원성 유전자들이 확인되었다.

This research was financially supported by the Ministry of Food and Drug Safety, Republic of Korea (23194MFDS016).

The authors have no conflict of interest to report.