Relationship between stx Genotype and Stx2 Expression Level in Shiga Toxin-Producing Escherichia coli O157 Strains

Abstract

SUMMARY:To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.Shiga toxin (Stx)-producing Escherichia coli (STEC) are emerging pathogens of significant clinical and public health concern that can cause symptoms ranging from mild to severe, including diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome, in humans (1). A STEC strain can produce any one of several Stx proteins or some combination of these. These proteins include Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g, and these toxins are encoded by the stx1, stx1c, stx1d, stx2, stx2c, stx2d, stx2e, stx2f, and stx2g genes, respectively. In Japan, O157 is the dominant serogroup among STEC of human origin, and most O157 isolates from humans produce Stx1, Stx2, Stx2c, or some combination of these 3 Stx variants (2-5). In our previous study, we demonstrated that each of 268 STEC O157 strains of human (211 strains) or cattle (57 strains) origin had 1 of the following 6 genotypes: stx1 alone (hereafter, stx1), stx2 alone (hereafter, stx2), stx2c alone (hereafter, stx2c), both stx1 and stx2 (hereafter, stx1/stx2), both stx1 and stx2c (hereafter, stx1/stx2c), or both stx2 and stx2c (hereafter, stx2/stx2c) (3). Of these 6 genotypes, only 5 (stx1 excluded) were detected among human isolates, and 5 genotypes (stx2/stx2c excluded) were detected among cattle isolates.Siegler et al. and Boerlin et al. showed that enteric infection by Stx2-producing E. coli was more likely to cause severe human disease than infection by organisms that produce only Stx1 (6,7). Furthermore, most STEC O157 strains carrying the stx2 gene (i.e., stx1/stx2, stx2, and stx2/stx2c) are more virulent than strains that do not carry the stx2 gene (i.e., stx1, stx1/stx2c, and stx2c) (2-5); therefore, it is reasonable to speculate that the severity of STEC O157-related diseases in humans may be a...

Virulence Characteristics and Antimicrobial Resistance Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Humans in South Africa: 2006–2013

“…The proportion of individual STEC isolates that carried both stx2c and stx2d concurrently was higher compared to the results of two studies, which previously reported single strains that possess both subtypes [12,49]. The presence of both stx2c and/or stx2d subtypes in a STEC isolate has been associated with human disease, varying from mild to bloody diarrhea and HUS [12,49,50,51,52].…”

Core and Accessory Genome Comparison of Australian and International Strains of O157 Shiga Toxin-Producing Escherichia coli

“…It has previously been reported that the United Kingdom 2015 outbreak was associated with high pathogenicity, and proposed that this was caused by the emergence of a clone that evolved from a STEC O157:H7 stx- negative ancestor after it acquired a bacteriophage encoding Shiga stx2a , which in turn had evolved from a stx2c progenitor ( Byrne et al, 2018 ). The Stx2a subtype has been reported to be most commonly associated with severe disease in humans, while Stx2c and Stx1a much less ( Ostroff et al, 1989 ; Boerlin et al, 1999 ; Orth et al, 2007 ; Kawano et al, 2012 ; Marejková et al, 2013 ; Brandal et al, 2015 ; Legros et al, 2018 ).…”

Augmenting the Detection of Verotoxin-Producing Escherichia coli Through use of Fluorescently-labeled DNA Oligonucleotides

“…VT2c, compared to 2a, is statistically associated with HUS although strains tested have been reported to produce less toxin and patients had a lower risk of developing HUS (Kawano et al, 2012;Friedrich et al, 2002). It is also frequently produced in combination with other VT subtypes and associated with eae (Orth et al, 2007).…”

“…VT2a has been found to be significantly associated with HUS and eae genes (a set of virulence factors involved in colonization of VTEC) and produced significantly higher amounts of toxin compared to other VT2 subtypes (Kawano et al, 2012;Beutin et al, 2004;Friedrich et al, 2002).…”

“…Increased amounts of VT de Sablet et al, 2008 Toxin Subtype Higher vtx mRNA and VT levels for VT2a compared to VT2c, even when the same serotype Kawano et al, 2012 The vtx genes encode the two subunits, A and B, sequentially in the phage DNA.…”