Preparation of Fluorescent Recombinant Shiga Toxin B Subunit and Its Application to Flow Cytometry.

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli (E. coli). Stx consists of one enzymatic A subunit and five B subunits (StxB) that are involved in binding. The StxB pentamer specifically recognizes a glycosphingolipid, globotriaosylceramide (Gb3), as a receptor; therefore, it can be used as a probe to detect Gb3. This chapter describes the preparation of recombinant Stx1B proteins using E. coli, their conjugation with fluorescent dyes, and their application for flow cytometry. The prepared fluorescent StxB proteins bound to cells of several lines, including the HeLa human cervix adenocarcinoma cell line and the THP-1 human monocytic leukemia cell line. Furthermore, the probe was useful for confirmation of several sphingolipid-deficient HeLa cell lines that were constructed using genome editing.

Biochemical Characterization of In vitro Reconstituted Biologically Active Recombinant Shiga Toxin.

BACKGROUND: Shiga toxins comprise a family of related proteins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in Haemolytic-Uremic Syndrome (HUS), up to 50% of HUS patients manifest some degree of renal failure and ~10% of such cases develop permanent renal failure or death. OBJECTIVE: In present research work production of biologically active rStx from non-toxic rStxA and rStxB subunits were established that can be used in many biomedical applications. METHODS: Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. To overcome this problem, the rStxA and rStxB protein were separately cloned and expressed in E. coli host and purified through affinity chromatography. GST pull-down assay was performed for interaction study between rStxA and pentameric rStxB. The affinity between A and B subunits of reconstituted recombinant Shiga toxin (AB5) was determined by SPR. The biological activity of the toxin was confirmed in Vero cells and mouse lethality assay. RESULTS: The yield of GST-StxA and His6X-StxB obtained after affinity chromatography was estimated to 2 and 5 mg/l, respectively. Samples analyzed in pull down assay revealed two bands of ~58 kDa (rStxA) and ~7.7 kDa (rStxB) on SDS-PAGE. Affinity was confirmed through SPR with KD of 0.85 pM. This rStx produced from 1:5 molar ratio found to be cytotoxic in Vero cell line and resulted lethality in mouse. CONCLUSIONS: Large scale production of rStx using the method can facilitate screening and evaluation of small molecule inhibitors for therapeutics development.

Screening for the presence of mcr-1/mcr-2 genes in Shiga toxin-producing Escherichia coli recovered from a major produce-production region in California.

The rapid spreading of polymyxin E (colistin) resistance among bacterial strains through the horizontally transmissible mcr-1 and mcr-2 plasmids has become a serious concern. The emergence of these genes in Shiga toxin-producing Escherichia coli (STEC), a group of human pathogenic bacteria was even more worrisome, urging us to investigate the prevalence of mcr genes among STEC isolates. A total of 1000 STEC isolates, recovered from livestock, wildlife, produce and other environmental sources in a major production region for leafy vegetables in California during 2006-2014, were screened by PCR for the presence of plasmid-borne mcr-1 and mcr-2. All isolates tested yielded negative results, indicating if any, the occurrence rate of mcr-1/mcr-2 among STEC was very low in this agricultural region. This study provides valuable information such as sample size needed and methodologies for future surveillance programs of antimicrobial resistance.

PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state.

In exposure to outer pressure, microorganisms are capable of entry into the Viable But Non-Culturable (VBNC) state, and thus survive under various elimination processing. The survival microorganisms may yield negative results on culturing, and cause false negative for this golden standard methodology. In this study, a novel PMA-LAMP assay on the detection of Enterohemorrhage E. coli and shiga toxins has been developed and evaluated, with further application on a number of food borne E. coli strains. LAMP primers were designed on the target of rfbe for Enterohemorrhage E. coli and stx1with stx2 for shiga toxins. Via specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on Enterohemorrhage E. coli and shiga toxins.

[The detection of strains of Esherichia coll producing Shiga toxin in populations of normal intestinal microbiota in children with functional disorders of gastrointestinal tract].

In intestinal ecosystem, interchange of genetic material between different types of bacteria and other representatives of family Enterobacteriaceae results in development of types of normal colibacillus with genetic characteristics of pathogenicity. This occurrence can be considered as a theoretical substantiation for labeling such strains as pathobionts. The polymerase chain reaction was implemented to analyze 96 strains of different types of Escherichia coli (with normal and weak zymogenic activity and hemolytic activity) isolated from children with functional disorders of gastrointestinal tract. The purpose was to detect presence of gens coding capacity of toxin production (six1, stx2). In intestinal biotope of children, circulation of strains of Escherichia coli producing shiga toxin having no relation to pathogenic group being representatives of normal indigenous microbiota. The presence of gens stx1 and stx2 in various biochemical types of Escherichia coli permits establishing fact of forming of reservoir of potential pathogenicity in non-pathogenic forms of Escherichia coli. The presence of gen (verotoxin 1) in genome of various types of Escherichia coli isolated from one single biotope testifies possible horizontal transmission of factors of pathogenicity in intestinal biotope.

Shiga toxins.

Shiga toxins are virulence factors produced by the bacteria Shigella dysenteriae and certain strains of Escherichia coli. There is currently no available treatment for disease caused by these toxin-producing bacteria, and understanding the biology of the Shiga toxins might be instrumental in addressing this issue. In target cells, the toxins efficiently inhibit protein synthesis by inactivating ribosomes, and they may induce signaling leading to apoptosis. To reach their cytoplasmic target, Shiga toxins are endocytosed and transported by a retrograde pathway to the endoplasmic reticulum, before the enzymatically active moiety is translocated to the cytosol. The toxins thereby serve as powerful tools to investigate mechanisms of intracellular transport. Although Shiga toxins are a serious threat to human health, the toxins may be exploited for medical purposes such as cancer therapy or imaging.

Microelectronic-sensing assay to detect presence of Verotoxins in human faecal samples.

AIMS: To develop a novel Vero cell assay that implements a real-time cell electronic sensing (RT-CES) system for the determination of the presence of verotoxin-producing Escherichia coli (VTEC). The assay overcomes the major drawbacks in conventional Vero cell assay, for example, labour-intensive and time-consuming. METHODS AND RESULTS: Cells were grown onto the surfaces of microelectronic sensors that are integrated into the bottom surfaces of the microtiter plate. Cellular viability was monitored in real-time and quantified based on changes in the sensor's electrical impedance. For cell viability measurement, the data generated on the RT-CES system correlated well with those obtained by the Vero cell assay for Verotoxins. To assess cytotoxicity, test cells growing on microelectronic sensors were treated with either supernatant from pure cultures, or stool samples. The specific neutralizing antibodies of VT1 and VT2 were used to identify specific toxins in the samples. CONCLUSIONS: The RT-CES assay provides a sensitive measurement comparable to conventional crystal violet assay. The assay has been successfully and specifically used to identify VTEC in human faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-CES assay significantly shortens the testing time from 48 to 72 h required by the crystal violet assay to only 15 h with automated operation.

Prevalence of virulence factors in Escherichia coli isolated from healthy animals and water sources in Brazil.

The aim of this work was to verify the presence of seven virulence factors (ST, LT, eae, stx(1), stx(2), INV and EAEC) among Escherichia coli strains isolated from healthy humans, bovines, chickens, sheep, pigs and goats, from two sewage treatment plants and from the Tietê River. We have found a high prevalence of eae, stx(1) and stx(2) in ruminants. The EAEC gene was only found in humans and sewage. No strains presented ST, LT or INV. BOX-PCR fingerprints revealed a high diversity among the strains analysed and a non-clonal origin of strains that presented the same virulence factors. Therefore, we concluded that ruminants may constitute an important reservoir of most diarrheagenic E. coli in Brazil, except for EAEC strains. These results emphasize the importance of the identification of the animal source of fecal contamination for the correct water risk assessment.

Shiga toxin-producing Escherichia coli (STEC) in Tennessee: surveillance and current clinical laboratory practices.

Since joining the Centers for Disease Control and Prevention's (CDC) Foodborne Diseases Active Surveillance Network (FoodNet) in 1999, Tennessee has conducted active surveillance for foodborne pathogens including Shiga toxin-producing E. coli (STEC). The number of STEC infections has increased in recent years in the United States, including Tennessee, due partly to changes in clinical laboratories practices including non-culture based testing methods. Despite increased reporting, STEC infections are likely under-recognized in Tennessee. A 2007 statewide laboratory survey indicated that less than half of clinical laboratories test for STEC on-site. Among these, only nine reported using non-culture based methods. Only one clinical laboratory reported simultaneous culture for STEC O157 and testing with an assay that detects Shiga toxins for non-O157 STEC as recommended by the CDC. Adoption of CDC recommendations coupled with timely and complete reporting will enhance public health surveillance, outbreak investigations and interventions to prevent STEC infection.

Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli O157 on cattle farms.

Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants. However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several transmission routes to humans are described, but cattle feces are the primary source from which both the food supply and the environment become contaminated with E. coli O157. In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 2007, vt-negative isolates were detected on 11.8% (8/68) of the positive farms. From these eight farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms. Further characterization revealed that all isolates carried the eaeA and hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE types and within the vt-negative strains, four different genotypes were identified, indicating that certain genetic clones are widespread over the cattle population.

[Bacteriological assessment of infectious diseases using a real-time PCR method in Tenri Hospital--focusing on diagnosis for atypical pneumonia and hemorrhagic enterocolitis].

In recent years, the detection of specific pathogens has been remarkably speedy with the development of new nucleic acid amplification methods such as real-time PCR, which is expected to be a great contribution to clinical diagnosis. Real-time PCR was introduced in our hospital, 2004 with the aim of detecting various pathogens. The system has been established and used for rapid bacteriological diagnosis for outpatient services in our hospital. This system can contribute greatly to achieving reliable and quick diagnosis, particularly for atypical pneumonia and hemorrhagic colitis caused by Escherichia coli. This system requires only 45 minutes on average for atypical pneumonia diagnosis, from receiving a specimen to the reporting of its results, which shortens the diagnosis time to about one-tenth of conventional methods. Survey on the type of initial dose in cases of Mycoplasma pneumoniae PCR positives shows that any administration of beta-lactam antibiotics, not effective to M. pneumoniae, has not been reported. Concerning the diagnosis of hemorrhagic colitis caused by Escherichia coli, it has been possible for us to report the results within one and a half hours, or within two hours including the legal notification of a third class infectious disease to a public health center. The SYBR Green Idetection system used in our hospital is superior to other detection systems comparing with versatility and cost-effectiveness. This report advocates that real-time PCR can contribute greatly to a rapid and cost-effective diagnostic system making full use of the characteristics of conventional bacteriological rapid-diagnostic methods, such as Gram staining and immuno-chromatography.

Síndrome hemolítico urémico asociado a infección por E. coli productor de toxina de shiga / Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome

E.coli productora de Toxina Shiga (STEC), también conocida como E.coli entero- hemorrágica (EHEC), provoca un amplio espectro de manifestaciones clínicas, ya sea en brote o en forma esporádica, que incluyen dolor abdominal, fiebre leve o ausente, con o sin vómitos, diarreas (sanguinolenta o no), y complicaciones extraintestinales como: síndrome hemolítico urémico (SHU) que se observa hasta en un 5-6 por ciento de niños infectados, y púrpura trombocitopénico (7 por ciento de adultos). EI principal factor de virulencia es la producción de una familia de moléculas denominada STX (Shiga toxin), de las cuales STX 1 y 2 son las más frecuentes y característica distintiva de estos E.coli. EI principal serogrupo involucrado en Chile es O157:H7 pero también se han aislado 026, 055, 02, 0117 y 06 (generalmente clasificadas como E. coli serogrupo clásico, no enterohemorrágico). Es fundamental para el clínico conocer la epidemiología, sintomatología y los exámenes que permitan un diagnóstico rápido para manejo terapéutico adecuado, y así evitar las complicaciones enunciadas anteriormente.

Shiga toxin producing E.coli (STEC), also known as enterohemorragic E.coli (EHEC), are responsible for a wide variety of clinical manifestations, both epidemic and sporadic. These include abdominal pain, no fever to mild fever, with or without vomits, diarrhea (bloody or not) and extraintestinal complications, such as haemolytic uremic syndrome in about 5 to 6 percent of children, and trombocitopenic purpura in 1 percent adults. The main virulence factor involved is the production of STX (Shiga toxin). In Chile there is marked prevalence of E.coli serogroup 0157 :H7 in these cases, although it has been associated also to E.coli 026, 055,02,0117 and 06, considered as classic serogroup (not enterohemorragic). It is of outmost importance for clinicians to be aware of symptoms and signs of this disease, as well as diagnostic methods that allow a prompt and adequate treatment, in order to avoid complications.

Prevention and treatment of enterohemorrhagic Escherichia coli infections in humans.

Infections with enterohemorrhagic Escherichia coli (EHEC) result in various clinical symptoms and outcomes ranging from watery or bloody diarrhea to the life-threatening hemolytic-uremic syndrome (HUS). Shiga toxins (Stxs) are supposed to play a major role in the pathogenesis of EHEC infections; however, the role of other putative virulence factors is not fully elucidated. So far, there is only supportive therapy available for the treatment of both EHEC-associated diarrhea and HUS. Antibiotic therapy for the treatment of EHEC-associated diarrhea is discussed. In recent years other therapeutic strategies have been developed, including Gb3 receptor analogues, that bind Stx in the gut or in the circulation, passive immunization with Stx-neutralizing monoclonal antibodies, or active immunization with Stx1 And Stx2 toxoids as a preventive procedure. These approaches have been demonstrated to be effective in animal models but clinical trials are lacking.

Presence of Shiga toxin-producing E. coli O157:H7 in a survey of wild artiodactyls.

This study was carried out to evaluate the role of wild artiodactyls as reservoirs of Escherichia coli O157:H7 for livestock and humans. Retroanal mucosal swabs samples from 206 red deer (Cervus elaphus), 20 roe deer (Capreolus capreolus), 6 fallow deer (Dama dama) and 11 mouflon (Ovis musimon), collected during the hunting season (autumn-winter) in South-western Spain, were screened. Samples were pre-enriched in modified buffered peptone water, concentrated by an immunomagnetic separation technique and cultured onto selective cefixime tellurite sorbitol MacConkey agar. Polymerase chain reaction (PCR) was used to detect the presence of genes coding O157 and H7 antigens and the virulence factors verocytotoxin, intimin and enterohaemolysin. Three E. coli O157:H7 isolates were obtained from red deer (1.5%). Two of them showed inability to ferment sorbitol and lack of beta-d-glucuronidase (GUD) activity, however, the other strain investigated was an atypical sorbitol-fermenting E. coli O157:H7 with GUD(+) activity. This is the first report pointing to red deer as a reservoir of E. coli O157:H7 in Spain.

Laboratory-confirmed non-O157 Shiga toxin-producing Escherichia coli--Connecticut, 2000-2005.

Shiga toxin-producing Escherichia coli (STEC) infection causes diarrhea that is often bloody and can result in potentially life-threatening hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 is the most common cause of STEC infection in the United States, producing 73,000 illnesses annually, according to the last estimate in 1999. Unlike O157, however, little is known about the incidence of non-O157 strains. Because STEC other than O157 are not commonly identified, the incidence, trends, and epidemiology of non-O157 STEC are not well understood. To assess trends in Shiga toxin enzyme immunoassay (Stx EIA) testing by local clinical laboratories, the Connecticut Department of Public Health (CTDPH) analyzed results of confirmatory testing conducted in the state laboratory during 2000--2005. The findings indicated that a total of 403 STEC infections were reported by clinical laboratories in Connecticut, including 207 identified as STEC by Stx EIA testing alone, and that the use of Stx EIA increased from 2000 to 2005. Use of Stx EIA without prompt culture confirmation can delay or prevent serotyping and subtyping of isolates and detection of both O157 and non-O157 STEC outbreaks. Public health authorities in all states should ensure that clinical laboratories forward Stx EIA-positive specimens to the state laboratory for isolation and identification of STEC, as recommended by the Association of Public Health Laboratories and CDC.

Quantitative detection of E. coli, E. coli O157 and other shiga toxin producing E. coli in water samples using a culture method combined with real-time PCR.

Recent water related outbreaks of shiga toxin producing E. coli O157 have resulted in increased attention of the water industry to this potentially deadly pathogen. Current methods to detect E. coli O157 and its virulence genes are laborious and time-consuming. Specificity, sensitivity and simple use of a real-time PCR method makes it an attractive alternative for the detection of STEC E. coli O157. This study describes the development and application of real-time PCR methods for the detection of E. coli O157, shiga toxin genes (Stx1 and Stx2) and E. coli. The specificity of the methods was confirmed by performing colony-PCR assays on characterized bacterial isolates, demonstrating the applicability of these assays as rapid tests to confirm the presence of E. coli or E. coli O157 colonies on culture plates. Sensitive culture-PCR methods were developed by combining culture enrichment with real-time PCR detection. This rapid method allowed detection of low concentrations of E. coli O157 in the presence of high concentrations of non-O157-E. coli (1:104). Culture-PCR methods were applied to 27 surface water and 4 wastewater samples. E. coli O157 and both Stx genes were detected in two wastewater samples, whereas only E. coli O157 was detected in two surface water samples. Culture-PCR methods were not influenced by matrix effects and also enabled quantitative (MPN) detection of E. coli in these samples.

Shiga toxin-producing Escherichia coli (STEC) O157 outbreak, The Netherlands, September-October 2005.

In September 2005, the first national food-related outbreak of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 was investigated in the Netherlands. A total of 21 laboratory-confirmed cases (including one secondary case), and another 11 probable cases (two primary and nine secondary cases) were reported in patients who became ill between 11 September and 10 October 2005. Preliminary investigation suggested consumption of a raw beef product, steak tartare (in the Netherlands also known as "filet americain"), and contact with other symptomatic persons as possible risk factors. A subsequent case-control study supported the hypothesis that steak tartare was the source of the outbreak (matched odds ratio (OR) 272, 95% confidence interval (CI) 3-23,211). Consumption of ready-to-eat vegetables was also associated with STEC O157 infection (matched OR 24, 95% CI 1.1-528), but was considered a less likely source, as only 40% of the cases were exposed. Samples of steak tartare collected from one chain of supermarkets where it is likely that most patients (67%) bought steak tartare, all tested negative for STEC O157. However, sampling was done three days after the date of symptom onset of the last reported case. Since 88% of the cases became ill within a two week period, point source contamination may explain these negative results. It is concluded that steak tartare was the most likely cause of the first national food-related outbreak of STEC O157 in the Netherlands.

Purification and characterization of cytolytic toxins produced by Aeromonas hydrophila and Aeromonas veronii biotype sobria strains.

Cytolytic toxins produced by Aeromonas hydrophila and Aeromonas veronii biotype sobria strains were partially purified from culture filtrates by two steps of purification: ammonium sulfate precipitation and hydrophobic chromatography using Phenyl-Sepharose CL-4B. Hemolytic activity was detected in one or two peaks in elution profile. Purified toxins were also cytotoxic to Vero and CHO cells. Moreover, these toxins revealed cytotonic activity to CHO cells.