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.     

Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.     

Evaluation of Enzyme-Labeled Oligonucleotide Probes to Identify Enterohaemorrhagic Escherichia coli

Alkaline phosphatase-conjugated oligonucleotide probes were developed to detect the gene coding for Vero toxin 1 (VT1) and Vero toxin 2 (VT2). Using these probes, 3 hr was enough to detect VT genes when suspicious colonies of enterohaemorrhagic Escherichia coli (EHEC) were obtained on an agar plate. The results of a hybridization test with 144 isolates of EHEC O157 and one isolate of Shigella dysenteriae Type 1 agreed exactly with the immunological detection, reversed passive latex agglutination (RPLA) test, of VTs in their culture supernatants. The sensitivity levels of these probes for the detection of VT genes were 100%. The specificity of these probes were also tested with a total of 1,002 strains of Escherichia coli other than EHEC and 8 strains of Shigella sp. other than Shigella dysenteriae Type 1; the results showed 100% specificity.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Detection and characterization of verocytotoxin-producing Escherichia coli (vtec) O157 and non-O157 in cattle at slaughter

Between September 2001 to June 2002, 145 samples of bovine caecal content were collected at slaughter for verocytotoxin-producing Escherichia coli (VTEC) serogroups O157 and non-O157 detection. For E. coli O157 the immunomagnetic-separation technique was performed. The enterohaemolytic phenotype was the target for non-O157 VTEC identification. The vero cell assay (VCA) was performed for toxic activity detection. The genomic sequence for VT1, VT2 and intimin (vt1, vt2, eae genes) were identified by PCR analysis. Eight VTEC O157 and eight non-O157 VTEC isolates were detected. VTEC O157, eae-positive strains were shed by 9.7% of feedlot cattle and by 2.5% of dairy cows. Non-O157 VTEC, eae-negative isolates were detected in the intestinal content of 12.5% dairy cows and of 2.1% feedlot cattle. VTEC-shedding cattle came from 18.1% of the farms included in the study. From cattle faeces, VTEC O91:H- (VT2-positive, eae-negative), responsible of human diarrhoeal disease in Europe, was recovered. Other VTEC serogroups identified in the present study were O74, O109, O110, O116, and O117.     

Verotoxin-producing Escherichia coli in culled beef cows grazing rangeland forages

The objective of this study was to assess prevalence of verotoxin-producing Escherichia coli (VTEC) in culled beef cows at the time of shipping to slaughter. Feces were collected from 82 cows on eight Nevada ranches during fall and winter (from September to January) after grazing rangeland forages. A random sample (n = 154) of potential VTEC isolates were tested for verotoxicity and were screened for the presence (polymerase chain reaction) and expression (VTEC-reversed passive latex agglutination assay) of the toxin genes (i.e., VT1 and VT2). Seventeen isolates from four ranches were VTEC. Of these, four had the VT1 gene, five had the VT2 gene, seven had both genes, and one did not have either gene despite its toxicity to Vero cells. Except for one isolate (i.e., untypeable that reacted with VT1-latex beads without having VT1 gene), the genotype and phenotype data of the VTEC isolates matched. Another isolate (O8:H- [nonmotile]) was verotoxic, but neither had nor expressed the toxin genes. Of the 17 isolates, four (from one cow) were O157:H7, 11 (from five cows on three ranches) were non-O157:H7 (two O8:H-, three O105:H-, three O116:H-, and three O141:H-), and two were untypeable. Because some of these VTEC serotypes (i.e., O8:H-, O141:H-, and O157:H7) are known to cause human illnesses, it is beneficial to identify VTEC-positive cows before slaughter. This is a critical step in any pre- or post-harvest strategy to minimize the risk of beef contamination with such pathogens.     

Detection of Verotoxigenic Escherichia coli by Magnetic Capture-Hybridization PCR

Magnetic capture-hybridization PCR (MCH-PCR) was used for the detection of 36 verotoxigenic (verotoxin [VT]-producing) Escherichia coli (VTEC), 5 VTEC reference, and 13 non-VTEC control cultures. The detection system employs biotin-labeled probes to capture the DNA segments that contain specific regions of the genes for VT1 and VT2 by DNA-DNA hybridization. The hybrids formed were isolated by streptavidin-coated magnetic beads which were collected by a magnetic particle separator and, subsequently, amplified directly by conventional PCR. The detection system was found to be specific for VTEC: no amplification was obtained from non-VTEC controls, whereas VTEC isolates tested positive for one or two specific PCR products. With 5, 7, or 10 h of enrichment, the limits of detection were 10³, 10², and 10⁰ CFU/ml, respectively, by agarose gel electrophoresis. Southern hybridization did not seem to improve the limit of the detection. When applied to food, MCH-PCR was capable of detecting 10⁰ CFU of VTEC per g of ground beef with 15 h of nonselective enrichment. The results of MCH-PCR for pure cultures of VT1- and/or VT2-producing E. coli cells were in total agreement with toxin production as measured by a VT enzyme-linked immunosorbent assay.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

A newly discovered verotoxin variant, VT2g, produced by bovine verocytotoxigenic Escherichia coli

A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H-.     

Examination of raw beef products for the presence of Vero cytotoxin producing Escherichia coli, particularly those of serogroup O157

Fifty-four of 310 (17%) samples of raw beef products contained Vero cytotoxin (VT)-producing Escherichia coli (VTEC) detected by DNA probes for the VT genes. VTEC strains examined in detail from a selection of the positive samples belonged to several O serogroups, some of which have been associated with human diarrhoea or haemolytic uraemic syndrome. Some of the strains possessed properties that may contribute to virulence in man. None of the food samples contained VT-producing E. coli O157 when tested by a combination of VT probe tests and colony immunoblotting with commercially available anti-O157 serum. Quantification of the immunoblotting technique indicated that O157 VTEC could be recovered from artificially-inoculated meat samples at a level of less than one organism per gram. Five of the food samples carried E. coli O157 strains that did not produce VT and differed in other properties from O157 VTEC.     

Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique

There has been no culture method of choice for detecting non-O157 Shiga toxin-producing Escherichia coli strains (STEC) because of their biochemical diversity The aim of this study was the assessment of verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. Stool cultures were performed on Tryptic Soy Broth and sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as Escherichia coli (E. coli) by BBL crystal. Further identifications were performed including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity, specificity of PCR were 100%. In comparative study between verotoxin assessment by Vero cells cytotoxicity and enterohemolytic activity, concordance positive results between both were 53 (91.4%). The most common serogroups of STEC were O157 (33%) and O26 (20%). From this study we can conclude that enterohemolysin production can be used as surrogate marker for STEC. The most rapid and promising approach for detection of STEC is by molecular method.     

Principles of some novel rapid dipstick methods for detection and characterization of verotoxigenic Escherichia coli

AIMS: The verotoxigenic Escherichia coli (VTEC) serotype most commonly associated with verotoxin (VT) production is O157:H7, but other serotypes have also been implicated in food-borne illness. These serotypes exhibit much greater genetic and biochemical diversity than E. coli O157:H7, making screening for all VTEC difficult. Here we describe development and testing of novel multi-analyte antibody-based dipstick methods for presumptive detection of VTEC cells and VTs, including non-O157 serotypes. METHODS AND RESULTS: The dipsticks are formatted as paddle-style and lateral flow devices. Test materials included raw milk, minced beef, apple juice and salami, spiked with VTEC. Prototype paddle dipsticks gave 47 of 48 E. coli O157-positive samples correct, and, simultaneously, 27 of 31 O26-positive samples correct, across the four food types. Prototype lateral flow dipsticks gave 12 of 12 E. coli O157-positive milk samples correct and, simultaneously, 28 of 28 positive VT samples correct. CONCLUSIONS: This work demonstrates that simple and rapid detection of more than one VTEC characteristic (toxin production and type, serogroup) is possible in a single dipstick test device, directly from a food enrichment culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of simple easy-to-use rapid methods for simultaneous detection and preliminary characterization of VTEC will enable the risk presented by all VTEC to be more thoroughly assessed (e.g. in surveillance studies, outbreak investigations).     

Susceptibility to verotoxin as a function of the cell cycle.

Infection with Verotoxin producing Escherichia coli (VTEC) has been implicated in hemolytic uremic syndrome, the leading cause of pediatric renal failure. Verotoxin (VT) binds to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4GlcCer Gb3) in susceptible cells. Gb3 is required for cytotoxicity and toxin-resistant cells deficient in Gb3 can be sensitized to VT cytotoxicity by incorporation of exogenous Gb3 into the cells. However, the absolute Gb3 content of cell lines does not necessarily correspond directly with the degree of sensitivity to VT. The present study demonstrates that susceptibility to VT is a function of cell growth and that stationary phase cells are resistant to VT. Using chemically synchronized Vero cells, we have also found a tenfold difference in susceptibility to VT during the cell cycle. Our experiments define a maximal sensitivity "window" of 1-2 hours from the G1/S boundary. This corresponds to increased VT binding without change in overall Gb3 content. Cell surface labelling indicated that cyclic turnover and exposure of Gb3 may be the critical parameter in determining VT sensitivity. Such changes during the cell cycle may also be of relevance in vivo in determining toxin pathology during VTEC infections and the physiology of plasma membrane Gb3.     

A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton

A highly sensitive, but simple and quantitative, cytotoxic assay method for the detection of toxic substances produced by red tide phytoplankton was developed by utilizing Vero cells which were the most resistant to seawater among the six cell lines tested. Heterocapsa circularisquama, which is known to be highly toxic to shellfish, showed cytotoxicity to Vero cells in a cell-density dependent manner when Vero cells were directly exposed to the cell suspension of H. circularisquama in seawater-based plankton culture medium, whereas Heterocapsa triquetra, which is morphologically similar to H. circularisquama but non-toxic to shellfish, showed no cytotoxic effect. Since the potent cytotoxicity was also detected in the cell-free culture supernatant of H. circularisquama, it was suggested that a certain cytotoxic substance is extracellularly secreted by H. circularisquama. Furthermore, by this direct exposure method, we found that Alexandrium fraterculus, Alexandrium tamiyavanichii, Alexandrium tamarense, and Alexandrium affine but not Alexandrium taylorii and Alexandrium catenella cause toxic effect on Vero cells with different extent depending on species. By gel-filtration and subsequent two cytotoxicity assays using Vero and mouse neuroblastoma cell line (Neuro-2a), we found that high molecular weight cytotoxic substance distinct from paralytic shellfish poisoning toxins is present in the aqueous extract of A. tamarense. These results suggest that our 96-well microplate cytotoxicity assay using Vero cells is useful not only as a primary screening assay for the detection of potential toxic activity of harmful phytoplankton but also as a quantitative routine toxicity assay for following the active substances during the extraction and purification processes.     

Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains

The Vero cell assay presently used for virulence testing of verotoxigenic Escherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was developed that measures endogenous lactate dehydrogenase (LDH) release from Vero or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp-2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strain also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cells. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell lines. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli showing positive LDH values. Similarly, RiboPrinter analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations from VTEC revealed severe lysis, vacuole formation and death in Vero cells and multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric cytotoxicity assay described here can provide quantitative data for determining the virulence potential of verotoxigenic E. coli in 12-16 h.     

Vero cytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain

Vero cytotoxin (VT)-producing Escherichia coli O157 (0157 VTEC) were isolated from a raw beefburger obtained from a retail source linked to a small community outbreak of 0157 VTEC infection in Wales. Strains from the meat and from seven of eight patients belonged to phage type 49 and were indistinguishable by their VT-type, plasmid content and hybridization with DNA of a VT-encoding phage from an 0157 VTEC strain. This first report of the isolation of 0157 VTEC from a beef product in Britain supports the view that there is a bovine reservoir for this organism.     

Rapid detection of diarrheagenic E. coli by real-time PCR

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.     

The detection of Vero cytotoxin-producing Escherichia coli and Shigella dysenteriae type 1 in faecal specimens using polymerase chain reaction gene amplification

Fifty consecutive faecal specimens received by the LEP were examined for the presence of Vero cytotoxin (VT) genes by polymerase chain reaction (PCR) gene amplification. Nineteen were positive by PCR and from 16 of these, VT positive Escherichia coli O157 were isolated. The remaining three samples were positive for VT genes by PCR but VTEC were not isolated. In a preliminary experiment, Shigella dysenteriae type 1 was isolated from a case of bloody diarrhoea following a positive amplification result.     

Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli O157: H 7

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.