Presence of Activatable Shiga Toxin Genotype ( stx _2d ) in Shiga Toxigenic Escherichia coli from Livestock Sources

Abstract

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10-to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx 2d . The stx 2 operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx 2 , stx 2c vha , stx 2c vhb , or stx 2d EH250 . Subsequently, the stx 2c vha and stx 2c vhb operons were screened for the absence of a PstI site in the stx 2A subunit gene, a restriction site polymorphism which is a predictive indicator for the stx 2d (activatable) genotype. Twelve STEC isolates carrying putative stx 2d operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx 2d . The complete nucleotide sequences of seven representative stx 2d operons were determined. Shiga toxin expression in stx 2d isolates was confirmed by immunoblotting. stx 2d isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages 1662a and 1720a carrying the stx 2d operons. RFLP analysis of bacteriophage genomic DNA revealed that 1662a and 1720a were highly related to each other; however, the DNA sequences of these two stx 2d operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.Shiga toxigenic Escherichia coli (STEC) isolates are important food-borne pathogens that exist as commensal bacteria of ruminant animals. Shiga toxins (Stxs) comprise an A subunit that carries the toxic function and a B-subunit pentamer that binds the toxin to the eukaryotic cell receptor (for a review, see reference 22). Studies have indicated that Stx type 2 (Stx2), encoded by the stx 2 operon, has an epidemiological relationship with the severe human disease conditions hemolytic-uremic syndrome and hemorrhagic colitis. In addition, toxins Stx2 and Stx2c, encoded by different genotypic subtypes of stx 2 , have also been associated with a greater severity of human disease. In contrast, the more recently described stx 2 genotype, stx 2d EH250 (26), is apparently of lesser clinical significance.The nomenclature designating the Stx2d EH250 subtype is complicated by the prior use of Stx2d to designate activatable Stx2 (16,18). Both Stx2d and Stx2d EH250 possess Ser291 and Glu297 residues in the Stx2A subunit toxin-coding region; however, only Stx2d is activatable. While STEC isolates carrying stx 2d EH250 are commonly found, particularly from sheep (8), very few reports describe activatable stx 2d genotype in STEC.Activatable stx 2d was originally detected in STEC strain B2F1. Two activatable operons, stx 2d1 and stx 2d2 , have been identified in the B2F1 (17). Prior to their designation as stx 2d1 and stx 2d2 , these opero...

Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature

“…One of the cornerstones of RFLP typing has been the absence of the PstI site (position 908 to 913), which has been used as an indicator of the presence of the mucus-activatable stx 2d subtype (10,14,21,26). However, the PstI site is also absent in 5 variants of stx 2a in E. coli (stx 2a -O113-CL-3, stx 2a -ONT-EBC217, stx 2a -O104-G5506, stx 2a -O8-VTB178, and stx 2a -ONT-pEHEC400) and in stx 2a -E-cloacae-95MV2, in two variants of stx 2c (stx 2c -O171-EBC287 and stx 2c -ONT-EBC289), in stx 2f , and in all four variants of subtype stx 2g .…”

A novel murine infection model for Shiga toxin–producing Escherichia coli

“…Recombinant Stx bacteriophages Φ1720aΔRz::cat and Φ1720aΔRz::cat Δstx2dactAB::kan were isolated from the newly modified genetically manipulated EHEC strains JB-13-06 and JB-13-28 (Supplemental Table 1), respectively, as previously described (66,70). Briefly, EHEC isolates were treated with mitomycin C (0.5 μg/ml) to induce the bacteriophage lytic cycle and production of phage.…”

“…Injection of Stx into mice results in the induction of proinflammatory cytokines, vascular damage, hemolysis, and renal damage (47)(48)(49)(50), but in contrast to piglet and rabbit infection models (23, 51-53), STEC strain originally isolated from ground beef (66). In preliminary screening of these C. rodentium lysogens, only C. rodentium (Φ1720a-02) produced significant amounts of toxin in vitro or caused systemic disease in mice (data not shown; see Figure 1).…”

Shiga toxins and stx phages: highly diverse entities

“…Furthermore, particular stx subtypes could affect the level of STEC shedding by cattle and consequently the risk of transmission to humans (Matthews et al, 2013). Although most stx subtypes have been detected in STEC strains isolated from cattle and beef products, some predominate amongst bovine STEC strains whereas others are rarely detected in such strains (Bertin et al, 2001;Brett et al, 2003a;Gobius et al, 2003;Beutin et al, 2007;Krüger et al, 2011). Examples of the latter include stx 1c , which has frequently been detected in STEC isolated from ovine faeces (Koch et al, 2001;Brett et al, 2003b), stx 2e , the common stx subtype in STEC strains responsible for oedema disease of swine (Linggood & Thompson, 1987;Weinstein et al, 1988), and stx 2f , detected in STEC strains isolated from the faeces of feral pigeons .…”