A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons

We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx(2f). This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx(2f) gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.     

Characterization of shiga toxin type 2 variant B-subunit in Escherichia coli strains from asymptomatic human carriers by PCR-RFLP

Subtyping of shiga toxin type 2 variant B-subunit in 35 non-O157 and two O157 strains isolated from 37 asymptomatic human carriers yielded two strains with stx2, 10 strains with stx2c and 24 strains with stx2d genes. One isolate harboured stx2 and stx2c. The high Stx2d prevalence in asymptomatic carriers was conspicuous and may indicate a reduced pathogenicity of these toxin variants. Therefore, in order to appraise a positive STEC laboratory result, the strain must be isolated in every case. Shiga toxin types and further virulence-associated factors have to be investigated.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

Effectiveness of Egg Yolk Antibody Against Shiga Toxin II Variant Toxicity In Vitro and In Vivo

We prepared anti-Shiga toxin II variant (Stx2e) and anti-Stx2e B antibodies in chicken egg yolk and investigated their effectiveness in vitro and in vivo. Both Egg yolk antibodies (IgY) reduced the cytotoxic effects of Stx2e to Vero cells. The protective efficacy of anti-Stx2e IgY and anti-Stx2e B IgY was investigated in clean Kunming mice, which were experimentally induced with Stx2e. Peritoneal injection of anti-Stx2e IgY and anti-Stx2e B IgY apparently reduced the lethal effect of Stx2e to mice. These results indicate that both anti-Stx2e IgY and anti-Stx2e B IgY could protect mice from Stx2e challenge. Therefore, anti-Stx2e IgY or anti-Stx2e B IgY might be considered as potential candidate for therapeutics for porcine edema disease.     

Passive protection of purified yolk immunoglobulin administered against Shiga toxin 1 in mouse models

Shiga toxins produced by Escherichia coli O157:H7 cause a wide spectrum of enteric diseases, such as lethal hemorrhagic colitis and hemolytic uremic syndrome. In this study, the B subunit protein of Shiga toxin type 1 (Stx1) was produced in the E. coli system, was further purified by Ni-column Affinity Chromatography method, and was then used as an immunogen to immunize laying hens for yolk immunoglobulin (IgY) production. Titers of IgY increased gradually with boosting vaccination and, finally, reached a level of 105, remaining steady over 1 year. Then the protective efficacy of IgY against Stx1 was evaluated by in vitro and in vivo experiments. It was shown that the anti-Stx1 IgY could effectively block the binding of Stx1 to the Hela cells and could protect BALB/c mice from toxin challenges. The data indicates the facility of using egg yolk IgY as a therapeutic intervention in cases of Shiga toxin intoxication.     

Transcriptional analysis of genes encoding Shiga toxin 2 and its variants in Escherichia coli

Six of 37 non-O157 Escherichia coli strains possessing Shiga toxin (Stx) 2 gene variant stx(2d) or stx(2e) secreted no detectable Stx. These isolates produced significantly less stx mRNA than Stx2d, Stx2e, Stx2c, or Stx2 secretors did. Standard screening procedures miss a significant subset of E. coli harboring stx(2) variants.     

Identification of a Shiga-toxin type I variant containing an IS1203-like element, from Shiga-toxin producing Escherichia coli O157:H7

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.     

Monoclonal antibody to shiga toxin 1, which blocks receptor binding and neutralizes cytotoxicity

A monoclonal antibody, 5-5B, which neutralizes Shiga toxin 1 (Stx1) cytotoxicity of Escherichia coli, was constructed. An epitope analysis indicated that Asn55 in Stx1 B subunit was an important residue. This result and our previous results using an anti-Stx2 monoclonal antibody indicate that the region around the cysteine residue of the disulfide bond might be important for the neutralization of Stx cytotoxicity, making it a potential vaccination candidate.     

单克隆抗体S2C4对2型志贺毒素及其亚型毒性的中和作用

纯化的2型志贺毒素(Shiga toxin 2,Stx2)经福尔马林脱毒后免疫BALB/c小鼠制备Stx2单克隆抗体,用体外中和试验对具有中和活性的阳性抗体克隆进行初筛,对所获得的中和抗体的重、轻链同种型及结合特异性进行鉴定,其中和保护作用通过体内、体外中和试验加以验证,最后,中和抗体对Stx2亚型Stx2c和Stx2vha的中和谱用体内中和试验验证．结果显示,12株抗Stx2的阳性抗体克隆中,只有1株具有中和活性,命名为S2C4,其重、轻链同种型为G₁/κ,其靶分子为Stx2的A亚单位,与Stx2的B亚单位或Stx1不结合．在体外中和试验中S2C4可有效中和Stx2对Vero细胞的杀伤作用,同样,S2C4可中和致死量的Stx2及其亚型Stx2c和Stx2vha对小鼠的毒性作用．该抗体有望成为治疗产志贺毒素大肠杆菌感染的候选分子．     

In vitro assessment of a chemically synthesized Shiga toxin receptor analog attached to chromosorb P (Synsorb Pk) as a specific absorbing agent of Shiga toxin 1 and 2.

A synthetic analog of Shiga toxin (Stx) receptor (Synsorb Pk) was quantitatively assessed to determine whether it can protect human renal adenocarcinoma cells (ACHN cells) from the cytotoxicity of Stx1 and Stx2 by coincubation experiments. Coincubation of 100 and 20 ng of Stxl and Stx2 with 50 mg of Synsorb Pk for 1 hr at 37 C in 1 ml of Eagle's Minimum Essential Medium supplemented with 1% (v/v) non-essential amino acid and 10% (v/v) fetal calf serum protected 50% of the cells from the cytotoxic effect. Chromosorb P, an inert matrix control, did not absorb the Stxs at all. Heat-treatment (boiled for 10 min) to Synsorb Pk caused a 50% decrease in Stx2-binding activity, but did not effect the Stx1 binding. Further, Stxs bound to Synsorb Pk could be demonstrated. When 20 mg of Synsorb Pk was coincubated for 30 min at 37 C in 1 ml of phosphate-buffered saline with 1 and 10 ng or more of Stx1 or Stx2, respectively, the toxins could be detected on the surface when the bound toxins on Synsorb Pk were used as the solid phase in enzyme immunoassay. The amount of 100 ng/ml of both Stxl and Stx2 appeared to saturate 20 mg/ml of Synsorb Pk after coincubating for 30 min at 37 C. While assessing the Stxs' binding activity to Synsorb Pk, it was demonstrated that Stxl had a higher affinity to Pk trisaccharide than Stx2. These observations provide useful information on the effectiveness of Synsorb Pk to trap and eliminate free Stxs produced in the gut of patients infected by Stx-producing Escherichia coli, and to prevent the progression of hemorrhagic colitis to hemolytic uremic syndrome.     

Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin-producing Escherichia coli

STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.     

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.     

Produce isolates of the Escherichia coli Ont:H52 serotype that carry both Shiga toxin 1 and stable toxin genes

Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.