Computational protocol: Complete Sequence of a Staphylococcus aureus Clonal Complex 81 Strain, the Dominant Lineage in Food Poisoning Outbreaks in Japan

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Protocol publication

[…] Staphylococcal food poisoning (SFP) is one of the common bacterial food poisonings contracted through the consumption of foods contaminated with toxic amounts of staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus (). We previously characterized isolates from SFP outbreaks in Japan and demonstrated that the clonal complex 81 (CC81) subtype 1 lineage is the major SFP-associated lineage (). In addition, other researchers reported that a similar lineage was frequently isolated in South Korea (, ). These results suggest that this bacterial group is widespread and frequently causes SFP outbreaks in far-east Asia. The major SE genotype of CC81 subtype 1 is sea seb seh sek seq. This lineage possesses sea-harboring ϕSa3mw2, which is a high-SEA-production-type prophage () and produces a large quantity of SEA in media and foods. Strain No. 10 is an isolate from an SFP outbreak in the 1990s in Tokyo, Japan (), and belongs to CC81 subtype 1 (). Genomic DNA was extracted with the QIAamp DNA purification kit (Qiagen GmbH, Hilden, Germany). A DNA library was prepared using the Nextera XT DNA library prep kit (Illumina, San Diego, CA). The 250-bp paired-end sequencing of No. 10 was carried out with the MiSeq platform (Illumina). The reads were mapped to the reference genome of S. aureus MW2 (GenBank accession no. BA000033) () by CLC Genomics Workbench (CLC bio, Arthus, Denmark), with an average 78-read mapping coverage. After the reads were assembled, contigs were aligned to MW2 with OSLay (). Gap closing was performed by Sanger sequencing. Only the gap within a cell wall protein corresponding to MW2416 remained, because this gene had a long G5-E repeat (), but we confirmed by PCR that this repeat number in No. 10 was the same as that in strain MW2.Strain No. 10 had a 2,764,435-bp chromosome containing 2,632 coding sequences (CDSs), 5 rRNA clusters, and 59 tRNAs and six staphylococcal enterotoxins/staphylococcal enterotoxin-like toxins (staphylococcal enterotoxin A [SEA], SEB, SEH, SEK, SEQ, and staphylococcal enterotoxin-like X [SElX]) genes. Compared with the closely related strain MW2 (CC1, a single-locus variant of CC81), No. 10 had an additional Staphylococcus pathogenicity island (SaPI [SaPIno10]) harboring seb and a putative truncated prophage (ϕSa2no10). Three SE genes, sea, sek, and seq, existed on a prophage similar to ϕSa3mw2. The seh and selx genes were located on a putative truncated transposon and chromosome, respectively. These locations in No. 10 were the same as those in MW2. Strain No. 10 lacked a staphylococcal cassette chromosome mec element (SCCmec), Panton-Valentine leukocidin (PVL)-associated prophage (ϕSa2mw2), and two enterotoxin (SEC and SEL)-associated SaPImw2, compared with MW2. Strain No. 10 possessed one plasmid, pNo10, whose length was 20,653 bp. This was classified as a type II plasmid with high similarity to the plasmids of MW2 and MSSA476, both of which were classified as CC1 (, ).Consequently, No. 10 had a genetic background similar to that of CC1, but there were some deletions, insertions, and mutations in some genes, such as enterotoxins, the toxin-antitoxin system, and serine proteases. These differences may contribute to the pathogenesis causing SFP. […]

Pipeline specifications

Software tools CLC Genomics Workbench, OSLay
Application De novo sequencing analysis
Organisms Staphylococcus aureus