Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Detection of Bacillus anthracis spore germination in vivo by bioluminescence imaging.

We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.

Identification and characterization of Shiga toxin type 2 variants in Escherichia coli isolates from animals, food, and humans.

There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx(2dact) that encodes the elastase recognition site. The presence of stx(2dact) was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx(2dact) gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx(1), two (P1332 and P1334) carried stx(1) and stx(2c), and one (CL-15) carried stx(2c). One isolate, P1130, harbored only stx(2dact). The Vero cell cytotoxicities of supernatants from P1130 and stx(1) deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.

Ricin crosses polarized human intestinal cells and intestines of ricin-gavaged mice without evident damage and then disseminates to mouse kidneys.

Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock.

Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli.

Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx(1) and/or stx(2), as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

Rapid detection of Shiga toxin-producing Escherichia coli by optical immunoassay.

In a multi-health center study, a new rapid optical immunoassay (OIA) for the detection of Shiga toxin types 1 and 2, the BioStar OIA SHIGATOX kit (Inverness Medical Professional Diagnostics, Inc.), was used to prospectively screen 742 fresh fecal samples for Shiga toxins in parallel with the Premier enterohemorrhagic Escherichia coli (EHEC) kit (Meridian BioScience, Inc.) with and without enrichment of the specimens by incubation in MacConkey broth. Additionally, 85 previously tested frozen fecal samples were assessed as described above. All positive immunoassay results were confirmed by the Vero cell cytotoxicity assay. A further modification of the screening procedure was evaluated on 470 of the prospectively screened specimens. Swabs of growth from conventionally plated stool culture media were subjected to the OIA SHIGATOX, and results were compared with those obtained with the Premier EHEC kit following broth enrichment. Overall, the OIA SHIGATOX kit was significantly more sensitive than the Premier EHEC kit on fresh direct stool specimens (sensitivities, 96.8% and 83.9%, respectively; P < 0.05). The two assays performed equally well with each other on frozen and broth-enriched samples. The colony sweep method used in conjunction with the OIA kit was somewhat more effective at detection of Shiga toxins from growth on agar than the overnight broth enrichment procedure used with the Premier EHEC assay (sensitivities, 100% and 92%, respectively; P < 0.09). Overall, the OIA SHIGATOX kit provided rapid, easy-to-interpret results and was highly effective at detection of Shiga toxin-producing E. coli in fecal samples and overnight cultures.

A plant-based oral vaccine to protect against systemic intoxication by Shiga toxin type 2.

Hemolytic uremic syndrome, the leading cause of kidney failure in children, often follows infection with enterohemorrhagic Escherichia coli and is mediated by the Shiga type toxins, particularly type 2 (Stx2), produced by such strains. The challenge in protecting against this life-threatening syndrome is to stimulate an immune response at the site of infection while also protecting against Shiga intoxication at distal sites such as the kidney. As one approach to meeting this challenge, we sought to develop and characterize a prototypic orally delivered, plant-based vaccine against Stx2, an AB5 toxin. First, we genetically inactivated the Stx2 active A subunit gene and then optimized both subunit genes for expression in plants. The toxoid genes were then transformed into the Nicotiana tabacum (tobacco) cell line NT-1 by Agrobacterium tumefaciens-mediated transformation. Toxoid expression was detected in NT-1 cell extracts, and the assembly of the holotoxoid was confirmed. Finally, mice were immunized by feeding with the toxoid-expressing NT-1 cells or by parenteral immunization followed by oral vaccination (prime-boost strategy). The immunized mice produced Stx2-specific mucosal IgA and Stx2-neutralizing serum IgG. The protective efficacy of these responses was assessed by challenging the immunized mice with E. coli O91:H21 strain B2F1, an isolate that produces an activatable variant of Stx2 (Stx2d) and is lethal to mice. The oral immunization fully protected mice from the challenge. Results of this study demonstrated that a plant-based oral vaccine can confer protection against lethal systemic intoxication.

Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge.

Shiga toxin type 1 (Stx1) and type 2 (Stx2) are produced by Escherichia coli O157:H7 and are responsible for the life-threatening sequela, the hemolytic uremic syndrome. Whether antisera to Stx1 or Stx2 are cross-neutralizing remains controversial, so we constructed genetic toxoids of Stx1 and Stx2 and evaluated them as vaccines. Antisera from mice immunized with a single toxoid type recognized and neutralized the homologous toxin but not the heterologous toxin. Furthermore, only mice immunized with Stx1 and Stx2 toxoids were protected against a lethal challenge of both toxins. We conclude that Stx1 and Stx2 are distinct antigens for mice.

Development of a hybrid Shiga holotoxoid vaccine to elicit heterologous protection against Shiga toxins types 1 and 2.

The hemolytic uremic syndrome is a life-threatening sequela that occurs after infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) or Shigella dysenteriae type 1, and Stx is responsible for initiating this syndrome. An STEC isolate can express Stx1, Stx2, or both, but antisera to Stx1 and Stx2 are not cross-neutralizing. To produce a single vaccine candidate against both toxins, we created a genetic toxoid that contained the enzymatically-inactivated StxA2 subunit and the native StxB1 subunit. We found that mice immunized with this hybrid holotoxoid, developed neutralizing anti-Stx1 and anti-Stx2 antibodies and survived challenge with 10 lethal doses of either or both toxins.

A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli.

We sought to develop a practical and representative model to study the interactions of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) with human intestinal tissue. For this purpose, human intestinal epithelial HCT-8 cells were cultured under low-shear microgravity conditions in a rotating cell culture system. After 10 days, layered cell aggregates, or 'organoids', developed. Three lines of evidence indicated that these organoids exhibited traits characteristic of normal tissue. First, the organoids expressed normal intestinal tissue markers in patterns that suggested greater cellular differentiation in the organoids than conventionally grown monolayers. Second, the organoids produced higher levels of intestinally expressed disaccharidases and alkaline phosphatase on a cell basis than did conventionally cultured monolayers. Third, HCT-8 organoid tissue developed microvilli and desmosomes characteristic of normal tissue, as revealed by electron microscopy. Because the low-shear microgravity condition is proposed by modelling studies to more closely approximate conditions in the intestinal microvilli, we also tested the impact of microgravity of bacterial growth and virulence gene expression. No influence on growth rates was observed but intimin expression by EHEC was elevated during culture in microgravity as compared with normal gravity. That the responses of HCT-8 organoids to infection with wild-type EPEC or EHEC under microgravitational conditions approximated infection of normal tissue was demonstrated by the classical appearance of the resultant attaching and effacing lesions. We concluded that the low shear microgravity environment promoted growth of intestinal cell organoids with greater differentiation than was seen in HCT-8 cells maintained in conventional tissue culture and provided a reduced gravity environment for study of bacterial-host cell interactions.

Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization.

The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin alpha.

Shiga toxin-producing Escherichia coli-associated kidney failure in a 40-year-old patient and late diagnosis by novel bacteriologic and toxin detection methods.

Infection with Shiga toxin-producing Escherichia coli (STEC) is the most common cause of kidney failure in children. High morbidity is also associated with infections in the elderly. We describe STEC-associated kidney failure in a 40-year-old patient, including the methods used to identify STEC a month after disease onset.

One of two copies of the gene for the activatable shiga toxin type 2d in Escherichia coli O91:H21 strain B2F1 is associated with an inducible bacteriophage.

Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx(2e) from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx(2d1)-containing lysogen was isolated from plaques on strain DH5alpha that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx(2d1)- and stx(2d2)-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.