Enterohemorrhagic <i>Escherichia coli</i> O157 subclade 8b strains in Chiba Prefecture, Japan, produced larger amounts of Shiga toxin 2 than strains in subclade 8a and other clades

<div><p>Enterohemorrhagic <i>Escherichia coli</i> O157 (O157) strains can be classified into clades (one of several phylogenetic groups) by single nucleotide polymorphisms (SNPs): these are clade 1, clade 2, clade 3, descendant and ancestral clades 4/5, clade 6, clade 7, clade 8, clade 9, and clade 12. Some recent studies showed that some O157 strains in clade 8 produced a larger amount of Shiga toxin (Stx) 2 than other strains. In this study, 1121 epidemiologically unlinked strains of O157 isolated in Chiba Prefecture, Japan were classified into clades during 1996–2014. Clade 8 strains were further classified into subclade 8a (67 strains) and subclade 8b (48 strains) using SNP analysis. In the absence of mitomycin C (MMC), subclade 8a strains in this study produced significantly greater amounts of Stx2 than subclade 8b strains. However, in the presence of MMC, the levels of Stx2 production in subclade 8b strains were significantly greater than subclade 8a strains. On the other hand, a recent study reported that the Stx2 production level in O157 strains was determined mainly by the subtypes of Stx2a phage (ϕStx2_α, β, γ, δ, ε, and ζ). Using O157 strains in this study, the Stx2a phages were classified into these subtypes. In this study, all strains of subclades 8a and 8b carried ϕStx2a_γ and ϕStx2a_δ, respectively. Some strains in clade 6 also carried ϕStx2a_δ. In the presence of MMC, subclade 8b strains produced significantly greater amounts of Stx2 than clade 6 strains carrying ϕStx2_δ. In this study, we propose that Stx2 production in subclade 8b strains in the presence of MMC might be enhanced due to genetic factors other than ϕStx2_δ.</p></div

The levels of Stx2 production in subclade 8a and 8b strains.

<p>The Stx2 titers of the subclade 8a and 8b strains in this study in the presence and absence of MCC are shown using the box-and-whisker plot. The bottoms of the lower bars show the minimum of Stx2 titers. The blue boxes show the Stx2 titers from the 25th percentile to the median. The orange boxes show the Stx2 titers from the median to the 75th percentile. The tops of the upper bars show the maximum of Stx2 titers. MMC + and MMC—indicate O157 strains that were treated and non-treated with MMC, respectively.</p

MLPT reconstructed from SNP data of clade 8 strains analyzed using the SNP set in this study.

<p>The dashed line marks branches with Stx2a phage subtypes. Red bars (A, C, E and G) indicate branches with multiple strains with both <i>stx2a</i> and <i>stx2c</i> genes. Red arrows indicate strains with both <i>stx2a</i> and <i>stx2c</i> genes. Black bars (B, D, F, H and I) indicate branches with multiple strains with only <i>stx2a</i>. Black arrows indicate strains with <i>stx</i>2a. The green arrow indicates a strain with a Stx2a phage that could not be typed.</p

The levels of Stx2 production in O157 strains carrying ϕStx2a_γ or ϕStx2a_δ with MMC treatment.

<p>The Stx2 titers in O157 strains carrying (A) ϕStx2a_γ or (B) ϕStx2a_δ in the presence of MCC are shown using the box-and-whisker plot. The bottoms of the lower bars show the minimum of Stx2 titers. The blue boxes show the Stx2 titers from the 25th percentile to the median. The orange boxes show the Stx2 titers from the median to the 75th percentile. The tops of the upper bars show the maximum of Stx2 titers.</p