Complete Genome Sequence of an Enterohemorrhagic <named-content content-type='genus-species'>Escherichia coli</named-content> O111:H8 Strain Recovered from a Large Outbreak in Japan Associated with Consumption of Raw Beef

We present the complete genome sequence of an enterohemorrhagic Escherichia coli O111:H8 strain. This strain was isolated from a hemolytic-uremic syndrome patient and was responsible for a large outbreak associated with the consumption of raw beef in 2011. Enterohemorrhagic Escherichia coli (EHEC) is a serious etiological agent of outbreaks of foodborne illness worldwide. More than 3,000 cases of EHEC infection are reported annually in Japan (1). In 2011, a large outbreak due to the consumption of raw beef was reported. A total of 181 patients, including 21 patients with acute encephalopathy (AE) and five deaths among 34 patients with hemolytic-uremic syndrome (HUS), were recorded (2, 3). EHEC O111:H8 and O157:H7 were isolated from the patients; however, the Shiga toxin 2a gene (stx2)-positive EHEC O111:H8 strain was the primary cause of the severe infection, according to serological testing of the patients (4). To reveal the characteristics of the strain, we determined its complete genome sequence, as the complete genome of the stx2-positive EHEC O111 strain has not been reported. EHEC O111:H8 110512 was isolated as one of a few colonies grown on deoxycholate-hydrogen sulfide-lactose agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) from the fecal sample of a 6-year-old boy with HUS and AE. The strain was stored in 0.5% NaCl casein peptone medium (Kohjin Bio Co., Ltd., Saitama, Japan) until use. For DNA extraction, a single colony was inoculated into buffered peptone water (Nissui). The overnight culture was subjected to genomic DNA extraction by a DNeasy blood and tissue kit (Qiagen, Venlo, Netherlands). The genomic DNA libraries were prepared using a Nextera DNA sample prep kit (Epicentre Biotechnologies, Madison, WI, USA), followed by paired-end sequencing (2 150-mer) with a MiSeq instrument (Illumina, San Diego, CA). Additionally, the strain was sequenced on a PacBio RS II instrument (Pacific Biosciences, Menlo Park, CA). The genomic DNA was fragmented prior to PacBio sequencing using the Megaruptor (Diagenode, Denville, NJ). PacBio sequencing was performed by using the SMRTbell template prep kit 1.0 and polymerase binding kit P6 after size selection using BluePippin (Sage Science, Beverly, MA) with a cutoff value of 20 kb. In bioinformatic analysis, default parameters were used for all software. The complete genome of the strain was obtained by using both PacBio and Illumina reads. PacBio reads were assembled using the Hierarchical Genome Assembly Process version 3 in SMRT Analysis software (5). The minimum seed length for the assembly was 6,000. Short plasmids ( 10 kb) were assembled using A5 MiSeq software version 20140604 with Illumina short reads (6). The tentative circular contigs were subjected to error correction in three iterations using Pilon version 1.18 with default parameters and Citation Sekizuka T, Lee K, Kimata K, Isobe J, Kuroda M, Iyoda S, Ohnishi M, Sata T, Watahiki M. 2019. Complete genome sequence of an enterohemorrhagic Escherichia coli O111:H8 strain recovered from a large outbreak in Japan associated with consumption of raw beef. Microbiol Resour Announc 8:e00882-19. https://doi.org/10.1128/MRA.00882-19. Editor David A. Baltrus, University of Arizona Copyright © 2019 Sekizuka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Masanori Watahiki, masanori.watahiki01@pref.toyama. lg.jp. Received 29 July 2019 Accepted 30 August 2019 Published 3 October 2019 GENOME SEQUENCES