In August 2023, 74 symptomatic patients developed Shiga toxin-producing Escherichia coli (STEC) O157 infection after consuming raw horse meat processed at a meat shop in Yamagata, Japan. To investigate the outbreak, we conducted multilocus variable-number tandem-repeat analysis (MLVA), whole-genome sequencing (WGS), and a nationwide retrospective epidemiological survey. MLVA and WGS identified 165 genetically similar strains collected across Japan during 2022-2023. Epidemiological investigations revealed that 6/89 (6.9%) cases in 2022 and 49/76 (64.5%) cases in 2023 were linked to raw horse meat consumption from the shop. WGS of 147 strains revealed a central genomic cluster of 76 strains, while others differed by 1-8 single-nucleotide variants from this cluster. The central genomic cluster contained strains from 2022 and 2023 obtained from patients who consumed raw horse meat. Epidemiological links and clonality of O157 strains suggested continuous contamination at the shop since 2022. Indeed, the shop lacked proper hygiene management based on hazard analysis and critical control point principles and exhibited poor sanitary conditions; however, direct evidence of the causative facility was not obtained because the suspected contaminated raw horse meat could not be sampled. This preliminary investigation highlights the importance of strict hygiene management in facilities producing meat for raw consumption and demonstrates the value of combining nationwide molecular epidemiology with field investigations to identify STEC outbreak sources. IMPORTANCE:Investigations of Shiga toxin-producing Escherichia coli (STEC) foodborne outbreaks incorporating whole-genome sequencing (WGS) have not been widely conducted to date. This study demonstrated that the combination of multilocus variable-number tandem-repeat analysis, WGS, and nationwide surveillance allowed for the detection of a dispersed STEC O157 outbreak linked to raw horse meat. Simultaneously, the investigation suggested persistent and low-diversity contamination at a single meat shop over a 1-year period. This study highlights (i) how combining genomic analysis with epidemiological investigation can identify geographically widespread STEC cases, (ii) the risk posed by inadequate hygiene management in facilities producing meat for raw consumption, and (iii) the potential for STEC strains to persist with minimal genomic mutations in food or processing environments. These insights support targeted control measures such as improved sanitation, the implementation of hazard analysis and critical control point principles, and ongoing genomic surveillance to prevent similar outbreaks.
Commonly available alternative methods for the simple identification of Omphalotus japonicus using a beam reagent (5 w/v% potassium hydroxide ethanolic solution) were investigated. As an alternative to the beam reagent used for both the direct and extraction methods, a 5 w/v% aqueous solution of sodium sesquicarbonate, a common ingredient used in household detergents, was demonstrated to be capable of identifying Omphalotus japonicus. Furthermore, the concentration of the extract solution of thelephoric acid, which is the coloring molecule, was the same in an 80% ethanol aqueous solution as it was in anhydrous ethanol. This equivalence suggests that ethanol, specified as a disinfectant in the Japanese pharmacopoeia, can be used as an extraction solvent. These findings suggest that identification equivalent to the conventional identification method can be achieved even when using commonly available sodium sesquicarbonate and disinfectant ethanol.
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of serious gastrointestinal illness, including diarrhoea, haemorrhagic colitis and life-threatening haemolytic-uraemic syndrome. Although O157:H7 STEC strains are the most prevalent, the incidence of STEC infections caused by several other serotypes has recently increased. O103:H2 STEC is one of these major non-O157 STEC strains, but systematic whole-genome sequence (WGS) analyses have not yet been conducted. To gain a global phylogenetic overview of O103:H2 STEC based on WGSs, we analysed 2,701 WGSs of O103:H2 strains, including 193 sequenced in this study. Sequence type (ST)-based classification divided the O103:H2 strains into three distinct E. coli lineages. As the virulence marker genes of typical STECs (stx, eae and ehxA) were found only in the major O103:H2 lineage (n=2,658) comprising ST17 and its single- and double-locus variants, we performed a global phylogenetic analysis of the major lineage. This analysis revealed that this lineage was divided into five clades (C1-C5) and that C1 was the ancestral clade, C2 and C3 emerged from C1 and C4 and C5 emerged from C3. While stx2 genes were sporadically distributed in limited STEC O103:H2 strains, stx1a, eae and ehxA were highly conserved throughout the entire STEC O103:H2 lineage. However, through a detailed comparison of seven closed genomes of STEC strains, covering the five clades and including four obtained in this study, we found marked variation in the genetic elements encoding the virulence genes (Stx1a phage, the locus of enterocyte effacement (LEE) and the virulence plasmid), such as rearrangement in the LEE accessory region, a shift in the integration sites of the Stx1a phage due to the replacement of the integrase gene-containing genomic segments, the replacement of the virulence plasmid and the gain and loss of virulence-related genes in the virulence plasmid. Overall, this study highlights the current global population structure of O103:H2 strains and provides evolutionary insights into the variation in virulence determinants within STEC O103:H2, which is relatively understudied among the major STEC lineages.
