Genome sequence of Escherichia coli NCCP15653, a group D strain isolated from a diarrhea patient
- Min-Jung Kwak†1,
- Myung-Soo Kim†1,
- Soon-Kyeong Kwon1,
- Seung-Hak Cho2 and
- Jihyun F. Kim1Email authorView ORCID ID profile
© Kwak et al. 2016
Received: 18 November 2015
Accepted: 4 January 2016
Published: 23 February 2016
Pathogenic strains in Escherichia coli can be divided into several pathotypes according to their virulence features. Among them, uropathogenic E. coli causes most of the urinary tract infections and has a genotype distinct from other virulent strains of E. coli. In this study, we sequenced and analyzed the genome of E. coli NCCP15653 isolated from the feces of a diarrhea patient in 2007 in South Korea.
A phylogenetic tree based on MLST showed that NCCP15653 belongs to the D group of E. coli and located in the lineage containing strains ST2747, UMN026 and 042. In the genome of NCCP15653, genes encoding major virulence factors of uropathogenic E. coli were detected. They include type I fimbriae, hemin uptake proteins, iron/manganese transport proteins, yersiniabactin siderophore proteins, type VI secretion proteins, and hemolysin. On the other hand, genes encoding AslA, OmpA, and the K1 capsule, which are virulence factors associated with invasion of neonatal meningitis-causing E. coli, were also present, while a gene encoding CNF-1 protein, which is a cytotoxic necrotizing factor 1, was not detected.
Through the genome analysis of NCCP15653, we report an example of a genome of chimeric pathogenic properties. The gene content of NCCP15653, a group D strain, demonstrates that it could be both uropathogenic E. coli and neonatal meningitis-causing E. coli. Our results suggest the dynamic nature of plastic genomes in pathogenic strains of E. coli.
KeywordsExtraintestinal E. coli UPEC NMEC Cystitis Pyelonephritis
Escherichia coli can be divided into commensal and pathogenic strains. Commensal E. coli is a member of the normal flora of animal intestine and other body sites, but pathogenic strains of E. coli cause several health problems. Many E. coli strains can cause diarrhea, but not serious . However, some pathogenic stains such as E. coli O104:H4 that caused the German outbreak in 2011 may be fatal . According to the virulence factors and phenotypes, pathogenic E. coli strains can be classified into enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), uropathogenic E. coli (UPEC), and E. coli that causes neonatal meningitis (NMEC) [3–8]. Among them, UPEC and NMEC are extraintestinal pathogenic E. coli (ExPEC), and most of the urinary tract infections (UTIs) are caused by UPEC strains . The urinary tract is a harsh environment because of continuous urine excretion, antibacterial factors, and strong immune system, and these features of urinary tract can make UPEC possible to have genotypes distinct to other pathogenic strains . In the urinary tract, it needs adhesion to urinary epithelial cells, several resistance factors against the antibacterial factors and host immune systems, and iron-acquisition systems to obtain iron, which is limited in the urinary tract. UPEC causes the infection in the bladder and sometimes in the kidneys through entering the ureters from the bladder to trigger symptoms such as cystitis and pyelonephritis, and even bacteremia and sepsis through entering the bloodstream . In this study, we sequenced and analyzed the genome of pathogenic E.coli strain NCCP15653 isolated from the feces of a patient suffering from diarrhea.
Bacteria and DNA isolation
E. coli strain NCCP15653 was isolated from the feces of a Korean patient with the diarrhea symptom in 2007. This strain was deposited at the National Culture Collection for Pathogens in Korea National Institute of Health (KNIH) and its accession number is NCCP15653. Genomic DNA was extracted using chemical and enzymatic methods as described in Molecular cloning, a laboratory manual .
Genome sequencing, de novo assembly and annotation
For the genome sequencing of NCCP15653, Genome Analyzer IIx of the Illumina platform at the Biomedical Genomics Research Center of the Korea Research Institute of Bioscience and Biotechnology was used and 18,521,148 of raw sequencing reads with 76-bp of average read length were generated from a 500-bp paired-end library. The sequencing reads were imported into CLC Genomics Workbench version 5.1 (CLC bio, Qiagen, Netherlands) with the parameters of 400–700 of paired-end distance and 1.5–1.7 version of Illumina quality score. Trimming of the imported reads was performed with the parameters of 0.01 quality score, none of the ambiguous nucleotide, and 70-bp of minimum read length. De novo assembly of 13,864,337 high-quality reads were conducted using CLC Genomics Workbench with the parameters of similarity fraction of 1.0, length fraction of 0.5, and minimum contig length of 500 bp. SSPACE  was used for scaffolding and IMAGE  was used for automatic gap filling. Manual contig extension and gap filling were performed with CLC Genomics Workbench. Structural gene prediction was accomplished with Glimmer3 , and functional annotation of predicted genes was performed using the MicroScope database .
A phylogenetic tree based on multilocus sequence typing (MLST) was constructed with MEGA5 . Nucleotide sequences of seven MLST genes (adk adenylate kinase, fumC fumarate hydratase, gyrB DNA gyrase, icd isocitrate/isopropylmalate dehydrogenase, mdh malate dehydrogenase, purA adenylosuccinate dehydrogenase, recA ATP/GTP binding motif)  and Jukes-Cantor model were used for tree construction. To determine the serotype of NCCP15653 in silico, amino-acid sequences of the wzx and wzy gene for O-antigen and the fliC gene for H-antigen were used and the neighbor-joining trees were constructed with MEGA5. SerotypeFinder program  was also used for the analysis. Average nucleotide identity based on blast (ANIb) value was calculated using JSpecies . Calculation of the core genome was conducted with OrthoMCL (ver. 2.0.3)  with parameters of e-value ≤1e–5, identity ≥85 %, and coverage ≥80 % . Functional classification of the genes was conducted by BLASTP with the COG and subsystem databases. Prediction of phage sequences and clustered regularly interspaced short palindromic repeats (CRISPRs) was performed with PHAST  and CRISPRfinder , respectively. Detection of the virulence genes was conducted using BLAST software. Typing of the specific virulence genes were referenced to the virulence factors of pathogenic bacteria database (http://www.mgc.ac.cn/VFs/main.htm) .
E. coli NCCP15653 was maintained in pure culture at KNIH and genomic DNA was isolated from a single isolate. Possibilities for the contamination of other genomes and misassembly were checked through mapping reads to the contigs. The read mapping of the draft genome of NCCP15653 indicated that the distance between paired-end reads is in the range of expected size distribution and the coverage of the reads was consistent throughout the genome.
Results and discussion
General features of the E. coli NCCP15653 genome
Number of contigs
Total contig length (bp)
Fold coverage (x)
G + C content (%)
Number of protein coding genes
Number of predicted transfer RNAs
Number of predicted ribosomal RNAs
GenBank accession number
Comparison with other E. coli strains in group D
Comparison of the virulence genes in the pathogenic strains in group D suggests that they may be divided into three groups (Fig. 3). The first group includes CE10 and IAI39, which are NMEC and UPEC, respectively. The second has UMN026, a UPEC strain, NCCP15653, and ST2747, which was isolated from a patient with UTI, but its pathotype is not yet determined . The third group contains the EAEC strain 042 alone, which has a quite different gene content compared to those in the first and second groups. Strains in the first and second groups show similar gene contents. However, in the genomes of CE10 and IAI39, aec (tss) genes encoding the type VI secretion system were not detected, and in the genome of NCCP15653, biosynthetic genes for aerobactin siderophore and P fimbriae were not present.
NCCP15653 was isolated from the feces of a diarrhea patient. However, an MLST-based phylogenetic tree and ANIb values indicated that NCCP15653 belongs to the D group of E. coli and is a sister strain of ST2747. In addition, in the genome of NCCP15653, genes encoding UPEC-type virulence factors of were detected, and those included type I fimbriae, hemin uptake proteins, iron/manganese transport proteins, yersiniabactin siderophore proteins, type VI secretion proteins, and hemolysin. Moreover, NCCP15653 has genes associated with the invasion of NMEC, which include those for the K1 capsule and putative arylsulfatase. Genome analysis results of NCCP15653 will be useful for further research of genome dynamics in the pathogenic E. coli strains causing UTI.
Availability of supporting data
This whole genome shotgun project of NCCP15653 has been deposited at GenBank under the accession ATLY00000000.
JFK conceived, organized and supervised the project, interpreted the results, and edited the manuscript. SHC characterized the strains and maintained it in pure cultures. SKK prepared the high-quality genomic DNA and arranged the acquisition of sequence data. MJK and MSK performed the sequence assembly, gene prediction, gene annotation, analyzed the genome information, and drafted the manuscript. All authors read and approved the final manuscript.
The authors are thankful to Byung Kwon Kim, Ju Yeon Song, Seon-Young Kim, and the KRIBB sequencing team for technical assistance. This work was financially supported by the National Research Foundation of the Ministry of Science, ICT and Future Planning (NRF-2011-0017670 to J.F.K.) and Korea National Institute of Health (KNIH 4800-4845-300 to S.H.C.), Republic of Korea.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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