STX2

Abstract: A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (stx1) called stx1OX3 (GenBank accession no. Z36901) was developed. The PCR was used to investigate 148 Stx1-producing Escherichia coli strains from human patients (n 72), cattle (n 27), sheep (n 48), and a goat (n 1) for the presence of the stx1OX3 gene. The stx1OX3 gene was present in 38 Shiga toxin-producing E. coli (STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx1-positive ovine STEC O91 strains. The stx1OX3 gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated with stx1OX3 carrying STEC from sheep and humans. In contrast, Stx1-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the stx1OX3 gene. The stx1OX3 -negative strains belonged to 13 serotypes which were different from those of the stx1OX3 -positive STEC strains. Moreover, the stx1OX3 gene was not associated with STEC belonging to enterohemorrhagic E. coli (EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying the stx1OX3 gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory E. coli K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx2-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx1-encoding bacteriophage H-19B from EHEC O26. The production of Shiga toxins (verocytotoxins) Stx1 and Stx2 is associated with certain strains of Escherichia coli. Ruminant animals such as cattle, sheep, and goats are naturally colonized with Shiga-toxin-producing strains of E. coli (STEC) and shed these organisms with their feces into the environment. Humans can be infected with STEC by contaminated food and by direct transmission of bacteria, and some STEC types pathogenic for humans cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (16). Over 200 serotypes of E. coli are known to be associated with the production of Shiga toxins (16). Analysis of the genes encoding Stx1 and Stx2 revealed the existence of various stx subtypes which show differences in their nucleotide sequences. In some strains, the stx

Abstract: Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.