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Complete genome sequence of enteropathogenic Escherichia coli MFDS1019811 isolated from donut
Korean J. Microbiol. 2024;60(1):50-53
Published online March 31, 2024
© 2024 The Microbiological Society of Korea.

Jina Ha, Doo Won Seo, Woojung Lee, Eun Sook An, Seung Hwan Kim, and Soon Han Kim*

Food Microbiology Division, Ministry of Food and Drug Safety, Osong, Cheongju 28159, Republic of Korea
Correspondence to: *E-mail: lambndog@korea.kr;
Tel.: +82-43-719-4301; Fax: +82-43-719-4300
Received November 28, 2023; Revised March 15, 2024; Accepted March 25, 2024.
Abstract
Escherichia coli, a foodborne pathogen that causes food poisoning, is implicated in intestinal infections. This study presents the complete genome sequence of enteropathogenic E. coli strain MFDS1019811, isolated from donut sample in 2021, which triggered a foodborne disease outbreak in Osan, South Korea. The complete genome sequence of E. coli strain MFDS 1019811 comprised 5,077,766 bp length chromosomal DNA and a 50,011 bp plasmid, with GC contents of 50.67% and 50.02%, respectively. Gene prediction revealed 92 tRNAs, 22 rRNAs and 5,048, and 90 CDSs on the genome. Several virulence genes were identified including bundle-forming pili (bfp) and intimin (eae).
Keywords : Escherichia coli, complete genome, donut
Body

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).

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.

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.

적 요

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.

Acknowledgments

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

Conflict of Interest

The authors have no conflict of interest to report.

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