The Prevalence of Shiga Toxin-1 in Non-Shigella Dysenteriae Isolates Collected from Diarrhea Samples in Patients, Ahvaz, Iran.

BACKGROUND: Acute diarrhea is a major public health problem, particularly in developing countries. Shigellosis is one of the substantial causative agents of microbial dysentery and still has a remarkable prevalence, particularly in areas with poor hygienic infrastructures. The probable existence of the deadly Shiga toxin (Stx) protein in some Shigella strains would manifest life-threatening clinical symptoms of the infection. METHODS: The aim of this study was to determine the presence of Shigella toxin 1 (Stx1) in isolated from patients with diarrhea. Totally, 227 Shigella species, including 60 S. flexneri, 157 S. sonnei, and 10 S. boydii were collected from diarrheal patients in the tropical infectious diseases research center of Ahvaz, Iran, during 2013-2015. The isolates were collected mostly from the intensive care unit, infectious disease, and surgery settings. The isolates were identified, and the polymerase chain reaction (PCR) was performed to detect the stx gene. RESULTS: The results indicated that none of them encode the stx1 gene. CONCLUSION: Isolates of this study were not capable of stx1 encoding. Future investigations should consider the relations between other Shigella species and Shigella toxin in Iran.

Shiga-like toxin I exerts specific and potent anti-tumour efficacy against gastric cancer cell proliferation when driven by tumour-preferential Frizzled-7 promoter.

OBJECTIVES: Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. METHODS: The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. RESULTS: Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. CONCLUSION: Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.

Relative proportions of E. coli and Enterococcus spp. may be a good indicator of potential health risks associated with the use of roof harvested rainwater stored in tanks.

A total of 285 water samples were collected from 71 roof harvested rainwater tanks from four villages in different provinces over a two-year (2013-2014) period during the early (October to December) and late (January to March) rainy season. Water quality was evaluated based on Escherichia coli, faecal coliforms and Enterococcus spp. prevalence using the IDEXX Quanti-Tray quantification system. Real-Time PCR was used to analyse a subset of 168 samples for the presence of Shigella spp., Salmonella spp. and E. coli virulence genes (stx1, stx2 and eaeA). Escherichia coli were detected in 44.1% of the samples, Enterococcus spp. in 57.9% and faecal coliforms in 95.7%. The most prevalent E. coli concentrations in harvested rainwater were observed in 29.1% of samples and 22.5% for Enterococcus spp. and, were within 1-10 cfu/100 ml and 10-100 cfu/100 ml, respectively, whereas those for faecal coliforms (36.6%) were within 100-1000 cfu/100 ml. On average 16.8% of the samples had neither E. coli nor Enterococcus spp. detected, while 33.9% had only Enterococcus spp. and 23.7% had only E. coli. E. coli and Enterococcus spp. were detected together in 25.5% of the samples. Evaluation of samples for potential pathogenic bacteria showed all tested samples to be negative for the Shigella spp. ipaH gene, while five tested positive for Salmonella ipaB gene. None of the samples tested positive for the stx1 and stx2 genes, and only two tested positive for the eaeA gene. These findings are potentially useful in the development of a simplified risk assessment strategy based on the concentrations of indicator bacteria.

Distribution of the stx1 and stx2 genes in Escherichia coli isolated from milk cattle according to season, age, and production scale in southwestern region of Goiás, Brazil / Distribuição dos genes stx1 e stx2 em Escherichia coli isoladas de bovinos de leite de acordo com a estação, idade e escala de produção na região sudoeste de Goiás, Brasil

This study determined the distribution of stx1 and stx2 genes in Escherichia coli isolated from dairy herds with regard to animal age, season, and farm production-scale, and analyzed the phylogenetic distribution of the groups A, B1, B2, and D of 276 isolates of bovine feces Shiga toxin-producing E. coli (STEC). The stx1 profile was the most common, detected in 20.4% (202/990) of the isolates, followed by stx2 (4.54%, 45/990) and stx1+stx2 (2.92%, 29/990). The stx1 gene was detected more frequently in calves than in adult animals. In the dry season (winter), the presence of stx1+stx2 profile in cattle feces was higher than in the rainy season (summer), while no significant changes were observed between seasons for the stx1 and stx2 profiles. The most predominant phylogenetic groups in adult animals were B1, A, and D, while groups A and B1 prevailed in calves. Our data highlight the importance of identifying STEC reservoirs, since 7.5% of the tested isolates were positive for stx2, the main profile responsible for the hemolytic-uremic syndrome (HUS). Moreover, these microorganisms are adapted to survive even in hostile environments and can contaminate the food production chain, posing a significant risk to consumers of animal products.(AU)

Esse estudo determinou a distribuição dos genes stx1 e stx2 em Escherichia coli isolados de rebanhos leiteiros em relação a idade, estação e produção, e analisaram a distribuição filogenética dos grupos A, B1, B2 e D de 276 E. coli produtoras de toxina Shiga (STEC). O perfil stx1 foi mais comum, detectado em 20,4% (202/990) dos isolados, seguido de stx2 (4,54%, 45/990) e stx1+stx2 (2,92%, 29/990). O gene stx1 foi detectado mais frequentemente em bezerros que animais adultos. No período de seca (inverno), a presença do perfil stx1+stx2 nas fezes dos bovinos foi mais prevalente que no período chuvoso (verão), apesar de não haver diferença significativa entre estações para os perfis stx1 e stx2. Os grupos filogenéticos mais predominantes em animais adultos foram B1, A e D, enquanto grupos A e B2 prevaleceram em bezerros. Nossos dados enfatizam a importância de se detectar reservatórios de STEC já que 7,5% dos isolados testados foram positivos para stx2, o perfil mais prevalente em casos de síndrome hemolítica-urêmica. Ademais, esses microorganismos são adaptados à sobreviver em ambientes hostis e contaminam a cadeia alimentar, levando a risco significativo para consumidores de alimentos de origem animal.(AU)

Frizzled-7 promoter is highly active in tumors and promoter-driven Shiga-like toxin I inhibits hepatocellular carcinoma growth.

Frizzled-7 protein plays a significant role in the formation of several malignant tumors. Up regulation of the Frizzled-7 in cancer cell lines is associated with nuclear accumulation of wild-type ß-catenin from the Wnt/ß-catenin pathway which is frequently activated in tumors. To analyze activity of the Frizzled-7 promoter in tumor cells, we constructed two recombinant plasmid vectors in which the Frizzled-7 promoter was used to drive the expression of green fluorescent protein (GFP) and Shiga-like toxin I (Stx1) (pFZD7-GFP/Stx1) genes. The Frizzled-7 protein was found to be expressed in the cancer cell lines but not in the normal cell lines. The GFP expression was restricted to the cancer cell lines and xenografts in the BALB/C mice but not to normal cell lines. Moreover, cell proliferation and tumor growth decreased significantly after transfection with the pFZD7-Stx1. Results from this study will help determine a highly effective strategy for gene therapy of tumors.

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples.

Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx1, stx2, and rfbEO157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbEO157, and LD-PCR results prompted successful recovery of E. coli O157 (n = 25) and non-O157 STEC (n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid "mega"-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA.

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

The nitric oxide reductase of enterohaemorrhagic Escherichia coli plays an important role for the survival within macrophages.

In enterohaemorrhagic Escherichia coli (EHEC) O157, there are two types of anaerobic nitric oxide (NO) reductase genes, an intact gene (norV) and a 204 bp deletion gene (norVs). Epidemiological analysis has revealed that norV-type EHEC are more virulent than norVs-type EHEC. Thus, to reveal the role of NO reductase during EHEC infection, we constructed isogenic norV-type and norVs-type EHEC mutant strains. Under anaerobic conditions, the norV-type EHEC was protected from NO-mediated growth inhibition, while the norVs-type EHEC mutant strain was not, suggesting that NorV of EHEC was effective in the anaerobic detoxification. We then investigated the role of NO reductase within macrophages. The norV-type EHEC produced a lower NO level within macrophages compared with the norVs-type EHEC. Moreover, the norV-type EHEC resulted in higher levels of Shiga toxin 2 (Stx2) within macrophages compared with the norVs-type EHEC. Finally, the norV-type EHEC showed a better level of survival than the norVs-type EHEC. These data suggest that the intact norV gene plays an important role for the survival of EHEC within macrophages, and is a direct virulence determinant of EHEC.

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Public health approach to detection of non-O157 Shiga toxin-producing Escherichia coli: summary of two outbreaks and laboratory procedures.

Routine laboratory testing may not detect non-O157 Shiga toxin-producing Escherichia coli (STEC) reliably. Active clinical, epidemiological, environmental health, and laboratory collaboration probably influence successful detection and study of non-O157 STEC infection. We summarized two outbreak investigations in which such coordinated efforts identified non-O157 STEC disease and led to effective control measures. Outbreak 1 involved illness associated with consuming unpasteurized apple cider from a local orchard. Public health personnel were notified by a local hospital; stool specimens from ill persons contained O111 STEC. Outbreak 2 involved bloody diarrhoea at a correctional facility. Public health personnel were notified by the facility infection control officer; O45 STEC was the implicated agent. These reports highlight the ability of non-O157 STEC to cause outbreaks and demonstrate that a coordinated effort by clinicians, infection-control practitioners, clinical diagnostic laboratorians, and public health personnel can lead to effective identification, investigation, and prevention of non-O157 STEC disease.

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2 percent) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66 percent) were typical (escv+, bfp+) and 14 (34 percent) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90 percent) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.

Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.

Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli.

A novel chromosome-plasmid hybrid bioluminescent reporter system (C-P reporter system) utilizing Photorhabdus luminescens luxCDABE genes has been constructed to monitor the expression of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) in enterohemorrhagic Escherichia coli (EHEC) in real time. The luxCDABE genes of P. luminescens have been cloned and divided into a luxCDAB cassette and a luxE gene. A promoter-less luxE gene introduced downstream from stx1 and from stx2 on EHEC chromosomes in single copies, and other luxCDAB genes were expressed on a multicopy number expression plasmid into the same cells. These Stx1- and Stx2-bioluminescent reporter strains expressed bioluminescence into bacteria cells when the expression of the promoter-less luxE gene was expressed in response to the promoter activity of stx1 and stx2, respectively. The expression levels of bioluminescence were identical to the production levels of Stx1 and Stx2 in the Stx1- and Stx2-bioluminescent reporter strains, and these strains produced both Stxs at the same respective levels as those of the parent EHEC strains. Using these reporter strains, we examined the profiles of Stx1 and Stx2 expression in EHEC. We found that production of both Stx1 and Stx2 in EHEC was enhanced upon contact with intestinal epithelial cells and within macrophages. However, the expression profiles between Stx1 and Stx2 in EHEC were different from each other under these conditions. Thus, these results suggested that this C-P reporter system is useful for determining the gene expression profile of bacteria.

Rapid and sensitive detection of Shiga toxin-producing Escherichia coli directly from stool samples by real-time PCR in comparison to culture, enzyme immunoassay and Vero cell cytotoxicity assay.

BACKGROUND: From May until July 2011 a large outbreak of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) occurred in Germany. More than 800 patients suffered from hemolytic uremic syndrome (HUS), and 49 fatal cases were reported. Obviously, a mandatory requirement for such a clinical situation is the availability of rapid and reliable STEC tests from the investigating laboratory. The standard methods like enzyme immunoassay (EIA), vero cell cytotoxicity assay (VCA), and microbiological culture are, however, hampered by a lack of sensitivity and specificity unless a prior, time consuming broth enrichment step is employed. In order to acelerate the laboratory diagnosis, we evaluated an in-house real-time PCR assay for the detection of the Stx genes (stx1 and stx2) directly from stool specimens without the need of broth enrichment procedures. METHODS: 754 faecal samples were collected from 481 predominantly hospitalised patients with diarrhea from May 23 to June 10, 2011 at the Medical Laboratory Bremen, Germany. The samples were analysed with a direct stx real-time PCR and compared to EIA, VCA and culturing on enterohemolysin, ESBL, and CPS agar after broth enrichment. In addition, artificial samples (n = 12) from three official EHEC/STEC PCR quality proficiency panels (INSTAND, Germany, September 2006, September 2007, and April 2008) were analysed by real-time PCR only. RESULTS: The real-time PCR produced reliable, distinct melting profiles with characteristic peaks for the stx1 and stx2 PCR products. The quality proficiency panels revealed a detection limit of 10 CFU/PCR per reaction. 112, 86, 99, and 122 of 754 clinical samples were positive for culture, EIA, VCA, and real-time PCR, respectively. 121 of 122 PCR samples were positive only for stx2. Compared to culture as the gold standard, sensitivities of EIA, VCA, and real-time PCR were 76.8 %, 83.9%, and 96.4% and specificities were 99.4%, 99.2%, and 97.8%, respectively. CONCLUSIONS: The direct fecal stx real-time PCR proved superior to enrichment based VCA and EIA and can be recommended as a quick and sensitive tool for the early diagnosis of STEC infection in addition to microbiological culture.

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3.

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes.

Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.

Efficient antiproliferative and antiangiogenic effects on human ovarian cancer growth by gene transfer of attenuated mutants of Shiga-like toxin I.

To evaluate the potential effect of anticancer and antiangiogenesis of Stx1(W203F) and Stx1(R170H), two attenuated mutants of Shiga-like toxin I (Stx1), in cancer gene therapy. Antiproliferative effects of these Stx1 mutants were tested in human ovarian carcinoma cell line SKOV3 and human umbilical vein endothelial cells (HUVECs) in vitro. Effect of these Stx1 mutants on inducing cell death and cell cycle arrest was analyzed in SKOV3 cells. Influence of these Stx1 mutants on endothelial cell function was analyzed in HUVECs. In vivo therapeutic effect of these Stx1 mutants on SKOV3 was explored using xenograft models in nude mice. These Stx1 mutants can inhibit the growth of SKOV3 or HUVECs and this effect can be abrogated by antibody specific for Stx1. They caused considerable cell death of SKOV3 cells in 24 h; neither caspase activity nor DNA fragmentation was observed, and necrosis is the major mode of cell death. These Stx1 mutants can induce cell cycle arrest of SKOV3 cells in G(2)-M or S phase depending on the dosage of gene transfer. Furthermore, they significantly decreased migration and capillary tube formation of HUVECs at low dose. In vivo study showed that Stx1(W203F) but not Stx1(R170H) significantly suppressed transplanted SKOV3 tumor growth in nude mice model. Interestingly, the microvessel densities of tumor treated with Stx1(W203F) and Stx1(R170H) were significantly reduced. This study suggests that genes encoding attenuated Stx1 can be selected as good candidates for the gene therapy of ovarian carcinoma because of their antiproliferative and antiangiogenic effects.

Metabolic and genetic profiling of clinical O157 and non-O157 Shiga-toxin-producing Escherichia coli.

A collection of clinical Shiga-toxin-producing Escherichia coli (STEC) strains, mainly belonging to serotypes O26, O103, O111, O145 and O157, was characterised by a polyphasic approach including molecular serotyping, PCR-based detection of virulence factors (stx1, stx2, eae, EHEC-hlyA, saa, katP, espP), carbohydrate fermentation profiles using API50 tests and random amplification of polymorphic DNA (RAPD) fingerprinting. An RAPD protocol based on the combination of 2 primers resulted in sufficiently complex patterns enabling discrimination to the serotype level. Moreover, carbohydrate fermentation profiles obtained after evaluating up to 50 different carbohydrates led to separation of different STEC serotypes. Virulence typing results confirm the association of Shiga toxins and intimin subtypes with specific serotypes and clinical diagnosis. Clinical diagnosis of strains did not correlate with either RAPD profiles or carbohydrate fermentation patterns.

Screening petting zoo animals for the presence of potentially pathogenic Escherichia coli.

Several outbreaks of Escherichia coli O157 have been reported in petting zoos, resulting in hospitalization of many children. At present, no standard procedure has been adopted to monitor the presence of enterohemorrhagic E. coli (EHEC) or Shiga-toxin-producing E. coli (STEC) in petting zoo animals. Direct detection of these strains from rectal swabs of animals in petting zoos was developed and obviated the need to culture the organisms. DNA extracted from bacteria in the swabs was tested for the presence of wecA gene specific for E. coli by polymerase chain reaction (PCR). The wecA positive samples were further tested for Shiga-toxin genes stxl and stx2, and the intimin eae by multiplex PCR and for the presence of O157 and H7. Swabs (n=104) from 15 animal species in a petting zoo were tested; 7 goats and 3 cows were found to carry STEC. The method is rapid and convenient for monitoring potentially pathogenic E. coli in petting zoo animals.