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Complete genome sequence of enteropathogenic Escherichia coli MFDS1001074 isolated from food
Korean J. Microbiol. 2022;58(4):324-326
Published online December 31, 2022
© 2022 The Microbiological Society of Korea.

Jiyoung Park1†, Heeyeon Ha2†, Eunsu Ha2, Woojung Lee1, Eun Sook An1, Seung Hwan Kim1, Jinho Choi2*, and Soon Han Kim1*

1Food Microbiology Division, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea
2Sanigen Co., Ltd, Anyang 14059, Republic of Korea
Correspondence to: *(J. Choi) E-mail: cjh@sanigen.kr; Tel.: +82-10-3211-8406; Fax: +82-2-573-3134 / (S.H. Kim) E-mail: lambndog@korea.kr; Tel.: +82-43-719-4301; Fax: +82-43-719-4300
These authors contributed equally to this work.
Received October 14, 2022; Revised December 15, 2022; Accepted December 16, 2022.
Abstract
Virulent strains of Escherichia coli can cause gastroenteritis, urinary tract infections, neonatal meningitis, hemorrhagic colitis, and Crohn’s disease in humans. This study elucidates the complete genome sequence of Escherichia coli strain MFDS1001074 isolated in 2012 from the ham of a high school meals in Cheongju, South Korea. The complete genome sequence of Escherichia coli strain MFDS1001074 consisted of a 4,807,852-bp chromosome and a 59,536-bp plasmid, with 50.49% and 49.08% G + C content, respectively. Gene predictions revealed that this strain contained 4,836 coding sequences (CDSs). Especially biofilm formation (csgB), capsule biosynthesis (etp), endotoxin (gtrAB), and type III secretion system (espADG) genes were identified.
Keywords : Escherichia coli, complete genome, ham
Body

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that induces epithelial cell actin rearrangement, resulting in pedestal formation beneath adherent bacteria (Ochoa and Contreras, 2011). Its main mechanism is to introduce attaching-and-effacing (A/E) lesions on intestinal cells, characterized by microvillus destruction, intimate adherence of strains to the intestinal epithelium, pedestal formation, and aggregation of polarized actin at sites of bacterial attachment (Frankel et al., 1998). In this study, we present the complete genome sequence of an EPEC strain collected from Cheongju, South Korea, in 2012.

EPEC MFDS1001074 was detected and isolated in a food poisoning investigation managed by the Ministry of Food and Drug (MFDS, South Korea) after it was reported at a high school in Cheongju in 2012. This strain was detected in ham. After isolation, MFDS1001074 was incubated anaerobically in tryptic soy agar medium at 37°C for 24 h. The total genomic DNA of MFDS1001074 was extracted using the Genomic DNA Prep Kit (Bioneer). Genomic DNA was qualitatively and quantitatively measured using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Fisher Scientific) and a Qubit 3.0 Fluorometer (Invitrogen). Complete genome sequencing was performed using the Illumina MiSeq and Oxford Nanopore MinION platform. For MiSeq sequencing, library preparation was performed using the Nextera DNA Flex Library Prep kit (Illumina) according to the manufacturer’s instructions. The size of the libraries was confirmed using a Bioanalyzer 2100 (Agilent Technologies). Illumina sequencing was performed using MiSeq system on a paired-end library with MiSeq Reagent Kit v2 (300-cycles). After sequencing, the individual sequence reads were analyzed using FastQC-v.0.11.8. The Illumina sequencing yielded 8,705,702 reads, including 1,190 Mb, with 239.7× coverage. The sequencing quality values Q20 and Q30 were 93.14% and 88.6%, respectively. For nanopore sequencing, the Oxford Nanopore Rapid Barcoding Kit (Oxford Nanopore Technologies) was used to construct libraries. Nanopore sequencing data were basecalled on guppy_barcoder v6.0.1. The Nanopore sequencing yielded 24,306 reads, including 104 Mb, with 20.1× coverage (N50, 8,128 bp). The sequencing mean quality was 13.5. The complete genome sequence of MFDS1001074 was 4,867,388 bp with a GC content of 50.47% (Table 1).

Genome features of EPEC MFDS1001074
Genomic feature Chromosome Plasmid
Contig 1 1
Genome size (bp) 4,807,852 59,536
GC content (%) 50.49 49.08
Genes 4,686 100
Protein-coding genes (CDSs) 4,829 100
tRNA genes 95 0
rRNA genes 22 0


Illumina and nanopore sequencing data were processed and de novo assembled with Unicycler v0.4.8, using the Pathosystems Resource Integration Center (PATRIC) v3.6.12 web server; the genome was then annotated using RASTtk (Brettin et al., 2015). Virulence associated genes were predicted using the Virulence Factor Database (VFDB) (Liu et al., 2019) and PATRIC_VF (Snyder et al., 2007). A total of 4,793 genes were predicted. EPEC were defined as containing intimin (eae) and lacking Shiga toxins. Additionally, typical EPEC possessed a virulence plasmid known as EPEC adherence factor (EAF) plasmid including bfpA, perA, perB, and perC (Schmidt, 2010). However, atypical EPEC did not contain EAF plasmid (Schmidt, 2010). MFDS1001074 was detected eae and not detected EAF plasmid, suggesting that the strain was atypical EPEC. MFDS 1001074 was found to contain virulence associated genes, involved in biofilm formation (agaBC, csgB, gatBCZ, kbaY), capsule biosynthesis (etk, etp, gfcABDE), endotoxins (gtrAB), and a type III secretion system (cesD, cif, escCDFJNQRSTUV, espADG, map) (Table 2). The complete genome information will be useful for investigating potential possibilities for understanding foodborne pathogens and providing a genetic basis for a more detailed analysis of virulence factors.

VAGs of EPEC MFDS1001074
Classification Gene(s) Predicted function References
Biofilm formation agaBC Dihydroxyacetone phosphate synthesis from galactitol and galactosamine Domka et al. (2007)
csgB Curli fibres Antão et al. (2009)
gatBCZ Dihydroxyacetone phosphate synthesis from galactitol and galactosamine Domka et al. (2007)
kbaY Ketose–bis–phosphate aldolase, tagatose–bisphosphate aldolase; part of aga cluster for K1 transport Berlyn et al. (1998)
Capsule biosynthesis etk An inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly Ilan et al. (1999)
etp Phosphotyrosine–protein phosphatase and involved in capsule formation Vincent et al. (2000)
gfcABDE Unknown functions specific to group 4 capsule export Larson et al. (2021)
Endotoxin gtrA Synthesis of undecaprenyl phosphate-D-glucose (UndP-D-Glc) from UDP-D-Glc and UndP Zdorovenko et al. (2018)
gtrB Translocation of UndP-D-Glc through the cytoplasmic membrane Zdorovenko et al. (2018)
Type III secretion system (TTSS) espAD Translocator protein, involved in intimate adherence and regulatory elements, secreted by TTSS of the locus of enterocyte effacement (LEE). Pakbin et al. (2021)
espG Effector protein, involved in intimate adherence and regulatory elements, secreted by TTSS of the LEE Pakbin et al. (2021)
map Effector protein, involved in intimate adherence and regulatory elements, secreted by TTSS of the LEE Pakbin et al. (2021)


Nucleotide sequence accession numbers

Nucleotide sequence accession numbers. The complete genome sequence of Escherichia coli MFDS1001074 has been deposited at the NCBI GenBank database under the accession numbers CP106929 (chromosome, MFDS1001074), CP106930 (plasmid, pMFDS1001074). The strain has been deposited in the Korean Culture Collection for foodborne Pathogens under the strain number MFDS1001074.

적 요

대장균의 독성 균주는 인간에서 위장염, 요로 감염, 신생아 뇌수막염, 출혈성 대장염 및 크론병을 유발할 수 있다. 이 연구는 2012년 대한민국 청주의 한 고등학교 급식의 햄에서 분리된 Escherichia coli (MFDS1001074)의 유전체 분석을 수행했다. Escherichia coli MFDS1001074는 4,807,852 bp 길이의 chromosomal DNA와 59,536 bp 길이의 plasmid로 구성되었으며, G + C contents는 각각 50.49% 및 49.08%였다. 유전자 예측에 따르면 이 균주는 염색체에 4,836개의 CDS를 나타냈다. 특히, biofilm formation (csgB), capsule biosynthesis (etp), endotoxin (gtrAB), type III secretion system (espADG) 관련 유전자를 확인했다.

Acknowledgments

This research was financially supported by the Ministry of Food and Drug Safety, Republic of Korea (20161MFDS030, 21162MFDS027).

Conflict of Interest

There are no conflicts of interest to declare.

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