Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.     

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

Inhibitory action of telithromycin against Shiga toxin and endotoxin

Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent.     

Role of Shiga toxin 2 (Stx2)-binding protein,human serum amyloid P component (HuSAP), in Shiga toxin-producing Escherichia coli infections: assumption from in vitro and in vivo study using HuSAP and anti-Stx2 humanized monoclonal antibody TMA-15

Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections. Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP). Here, we report the role of HuSAP in STEC infections. HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice. By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP. These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP.     

The effect of probiotics and organic acids on Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157:H7

Probiotics are known to have an inhibitory effect against the growth of various foodborne pathogens, however, the specific role of probiotics in Shiga-toxin-producing Escherichia coli (STEC) virulence gene expression has not been well defined. Shiga toxins are members of a family of highly potent bacterial toxins and are the main virulence marker for STEC. Shiga toxins inhibit protein synthesis in eukaryotic cells and play a role in hemorrhagic colitis and hemolytic uremic syndrome. STEC possesses Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), both of which have A and B subunits. Although STEC containing both Stx1 and Stx2 has been isolated from patients with hemorrhagic colitis, Stx2 is more frequently associated with human disease complications. Thus, the effect of Lactobacillus, Pediococcus, and Bifidobacterium strains on stx2A expression levels in STEC was investigated. Lactic acid bacteria and bifidobacteria were isolated from farm animals, dairy, and human sources and included L. rhamnosus GG, L. curvatus, L. plantarum, L. jensenii, L. acidophilus, L. casei, L. reuteri, P. acidilactici, P. cerevisiae, P. pentosaceus, B. thermophilum, B. boum, B. suis and B. animalis. E. coli O157:H7 (EDL 933) was coincubated with sub-lethal concentrations of each probiotic strain. Following RNA extraction and cDNA synthesis, relative stx2A mRNA levels were determined according to a comparative critical threshold (Ct) real-time PCR. Data were normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the level of stx2A expression between treated and untreated STEC was compared. Observed for all probiotic strains tested, stx2A was down-regulated, when compared to the control culture. Probiotic production of organic acids, as demonstrated by a decrease in pH, influenced stx2A gene expression.     

The CI repressors of Shiga toxin-converting prophages are involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2

Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.     

Spread of a Distinct Stx2-Encoding Phage Prototype among Escherichia coli O104:H4 Strains from Outbreaks in Germany, Norway, and Georgia

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.     

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.     

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Comparison of Shiga toxin production by hemolytic-uremic syndrome-associated and bovine-associated Shiga toxin-producing Escherichia coli isolates

There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.     

Genetic typing of shiga toxin 2 variants of Escherichia coli by PCR-restriction fragment length polymorphism analysis

Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx(2) gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx(2) genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx(2) variants and STEC isolates carrying multiple stx(2) genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx(2) sequences with the same PvuII HaeIII HincII AccI type generally cluster together.     

Analysis of Twin-Arginine Translocation Pathway Homologue in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We investigated the relationship between expression of the O side chain of outer membrane lipopolysaccharide (LPS) and infection by a Shiga toxin 2 (Stx2)-converting phage in normal and benign strains of Escherichia coli. Of 19 wild-type E. coli strains isolated from the feces of healthy subjects, those with low-molecular-weight LPS showed markedly higher susceptibility to lytic and lysogenic infection by Stx2 phages than those with high-molecular-weight LPS. All lysogens produced infectious phage particles and Stx2. The Stx-negative E. coli O157:H7 strain ATCC43888 with an intact O side chain was found to be resistant to lysis by an Stx2 phage and lysogenic infection by a recombinant Stx2 phage, whereas a rfbE mutant deficient in the expression of the O side chain was readily infected by the phage and yielded stable lysogens. The evidence suggests that an O side chain deficiency leads to the creation of new pathotypes of Shiga toxin-producing E. coli (STEC) within the intestinal microflora.     

Escherichia coli serogroup O107/O117 lipopolysaccharide binds and neutralizes Shiga toxin 2

The AB(5) toxin Shiga toxin 2 (Stx2) has been implicated as a major virulence factor of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains in the progression of intestinal disease to more severe systemic complications. Here, we demonstrate that supernatant from a normal E. coli isolate, FI-29, neutralizes the effect of Stx2, but not the related Stx1, on Vero cells. Biochemical characterization of the neutralizing activity identified the lipopolysaccharide (LPS) of FI-29, a serogroup O107/O117 strain, as the toxin-neutralizing component. LPSs from FI-29 as well as from type strains E. coli O107 and E. coli O117 were able bind Stx2 but not Stx1, indicating that the mechanism of toxin neutralization may involve inhibition of the interaction between Stx2 and the Gb(3) receptor on Vero cells.     

Anisodamine inhibits shiga toxin type 2-mediated tumor necrosis factor-alpha production in vitro and in vivo

Cytokines, in particular tumor necrosis factor (TNF), appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study we examined the effect of anisodamine, a vasoactive drug, on TNF-alpha production in Shiga toxin type 2 (Stx2)-stimulated human monocytic cells in vitro and in Stx2-injected mice sera in vivo. Human monocytes and THP-1 cells were stimulated by Stx2 (1-100 ng/ml) with or without anisodamine addition (1-400 micrograms/ml). For in vivo evaluations, C57BL/6 mice were given a single intraperitoneal injection of anisodamine (6-50 mg/kg) or saline after intraperitoneal injection of Stx2 (50 ng/kg). The results showed that anisodamine suppressed Stx2-induced TNF-alpha production in a dose- and time-dependent manner. Anisodamine also suppressed Stx2-induced TNF-alpha mRNA expression. Further study showed that endogenous prostaglandin E2 may be involved in this inhibitory effect. In contrast to TNF-alpha mRNA, anisodamine at concentrations as high as 400 micrograms/ml did not decrease Stx2-induced IL-1 beta and IL-8 mRNA levels. In addition, anisodamine (> 50 micrograms/ml) increased Stx2-stimulated THP-1 cell viability. Levels of TNF-alpha in anisodamine-treated mice sera were significantly lower than those in the saline-treated group 1.5 and 24 hr after Stx2 injection. Anisodamine induced a lower percentage of death in Stx2-injected mice. Taken together, our results indicate that anisodamine has an important regulatory effect on Stx2-induced TNF-alpha production in vitro and in vivo. The present study suggested that this drug should be further investigated for its effects on Stx2-mediated diseases in humans.     

60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage

Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.     

Role of nitric oxide in tolerance to lipopolysaccharide in mice.

The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the febrile response, which is called endotoxin tolerance. We tested the hypothesis that nitric oxide (NO) arising from inducible NO synthase (iNOS) plays a role in endotoxin tolerance, using not only pharmacological trials but also genetically engineered mice. Body core temperature was measured by biotelemetry in mice treated with NG-monomethyl-L-arginine (L-NMMA, 40 mg/kg; a nonselective NO synthase inhibitor) or aminoguanidine (AG, 10 mg/kg; a selective iNOS inhibitor) and in mice deficient in the iNOS gene (iNOS KO) mice. Tolerance to LPS was induced by means of three consecutive LPS (100 microg/kg) intraperitoneal injections at 24-h intervals. In wild-type mice, we observed a significant reduction of the febrile response to repeated administration of LPS. Injection of L-NMMA and AG markedly enhanced the febrile response to LPS in tolerant animals. Conversely, iNOS-KO mice repeatedly injected with LPS did not become tolerant to the pyrogenic effect of LPS. These data are consistent with the notion that NO modulates LPS tolerance in mice and that iNOS isoform is involved in NO synthesis during LPS tolerance.     

Sensitive detection of Shiga Toxin 2 and some of its variants in environmental samples by a novel immuno-PCR assay

Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Comparison of Shiga Toxin Production by Hemolytic-Uremic Syndrome-Associated and Bovine-Associated Shiga Toxin-Producing Escherichia coli Isolates

There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.