A method of enhancing verocytotoxin production by Escherichia coli

The number of verocytotoxin producing Escherichia coli (VTEC) present in the faeces during an infection may be very low, making their detection difficult. We report a method for enhancing toxin production by VTEC using mitomycin C as an inducing agent with the aim of improving the detection of VTEC. In pure culture, mitomycin C enhanced toxin production up to 100-fold. When applied to mixed faecal culture, toxin could be detected in mitomycin C treated samples when standard cultures were negative and when substantially fewer verocytotoxin-producing bacteria were present. Use of this method may aid in the detection of VTEC and is appropriate for use in the routine diagnostic laboratory.     

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).     

Phenotypic and molecular characteristics of typical and atypical Escherichia coli O157, clinical and food isolates

Enrichment, colony isolation and confirmation are three general phases of a standard diagnostic method. E. coli O 157 (the main member of EHEC group) differs metabolically from other strains of E. coli in a number of ways. Most isolates are slow- or non-fermenters of sorbitol and lack the enzyme beta-glucuronidase (GUD). But, a variety of atypical strains of E. coli O157 (sorbitol-fermenting variants, nonmotile and GUD-positive) have been reported. The discovery of these atypical pathogenic strains brings into question the validity of testing for the pathogen only by biotyping. Using classical cultivation and immunomagnetic separation, we have isolated from food a few atypical E. coli O157 (sorbitol-fermenting strains, GUD positive, nonmotile O157 strain which does not agglutinate with O157 latex and does not produce Shiga toxin). On the other hand, non-O157 VTEC (O26 serotype) producing Shiga toxin was isolated from meat. Molecular markers of E. coli O157 and virulence-associated factors of strains with aberrant biochemical properties were studied by PCR. This method helped us in the final identification of isolates. Since it was suggested that the production of verotoxins (VT) is accompanied by the production of enterohemolysin (Ehly) such correlation has also been evaluated in respect to the collection of VTEC of human, animal and food origin.     

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.     

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.     

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.     

Verotoxin-producing Escherichia coli in sheep grazing an irrigated pasture or arid rangeland forages

Worldwide, verotoxin-producing Escherichia coli (VTEC) have been recognized as the cause of many sporadic cases or major outbreaks of human illnesses involving consumption of contaminated meat, especially beef. Although sheep products have not been linked to reported human illnesses, their role as a food safety risk factor should not be ignored. The objective of this study was to assess VTEC prevalence in two groups of ewes (20 each) grazing an irrigated pasture or arid range in a western United States environment (Nevada) over 1 year (summer of 1999 to summer of 2000). A random sample (n = 504) of potential VTEC isolates were tested for verotoxicity and were screened for the presence (polymerase chain reaction [PCR]) and expression (VTEC-reversed passive latex agglutination assay) of the toxin genes (i.e., VT1 and VT2). Forty-one VTEC isolates (16 having only the VT1 gene and 25 having both VT1 And VT2 genes) were detected in both groups of ewes. Except for seven isolates, the genotype and phenotype data matched. All the isolates (nonmotile [H-]) were non-O157:H7 VTEC (i.e., O91:H- [n = 25], O128:H- [n = 9], and untypeable ones [n = 7]). More infected ewes (nine versus three) and different VTEC strains were found in the irrigated pasture than in the arid range. Because our ewes were shedding two VTEC serotypes known to cause human illnesses, it is beneficial to identify VTEC-positive sheep before slaughter as an initial control point before entering the food chain.     

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin‐producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time‐consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non‐O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti‐O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time‐resolved fluorescence immunoassay (TRFIA). Copyright © 2002 John Wiley & Sons, Ltd.     

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.     

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.     

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.     

Ability of human sera to neutralise the activity of Vero cytotoxins VT1, VT2 and variant forms of VT2

Abstract Sera, from 17 patients with diarrhoea or haemolytic uraemic syndrome, and six healthy adults, were tested for neutralisation of Vero cytotoxins (VT). For all 17 patients there was evidence of infection with Escherichia coli O157. Sera from two controls but from none of the patients neutralised VT1, although two patients were infected by strains producing VT1 and VT2. Sera from all six controls and 14 patients neutralised VT2 derived from strains 933 and E32511, but not variant forms of VT2 derived from strains E32511, E57, B2F1 and H.1.8. This neutralising activity warrants further investigation, especially as many 0157 VTEC carry both VT2 and VT2 variant genes.     

Role of non-O157 VTEC

Non-O157 VTEC are typical Escherichia coli that differ only in their ability to produce verocytotoxins (VT). The transmission of VTEC is discussed in relation to the transmission of commensal E. coli. The emergence over the last few decades of a great variety of VTEC serotypes from healthy and diseased humans and animals is described. Particular attention is given to the distribution of the more important serogroups pathogenic for humans that have been described from around the world, particularly serogroups O26, O111, O128 and O103. The possible role of ruminants as reservoirs is discussed. The problems of laboratory diagnosis of non-O157 VTEC are considered and various laboratory methods are assessed. Evidence is presented that the particular E. coli serotypes now known to be VTEC were present in humans and animals many years ago, but have acquired the ability to produce VT and probably other virulence factors. Finally, predictions are made of the possible increase in problems associated with these emerging pathogens.     

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.     

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.     

Description of a novel intimin variant (type zeta) in the bovine O84:NM verotoxin-producing Escherichia coli strain 537/89 and the diagnostic value of intimin typing

Infections with verotoxin-producing Escherichia coli (VTEC) has resulted in increasing numbers of human illnesses annually. These illnesses usually result from the ability of VTEC to cause the attaching and effacing lesions (AE lesion). The AE phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. A key adhesion factor involved is the outer membrane protein intimin, encoded by the eae gene within the LEE. Intimin types alpha, beta, gamma, delta, and epsilon have been described previously. Each intimin represents distinct phylogenetic lineages of LEE-positive strains. A new intimin type zeta was identified in a VTEC strain of the serotype O84:NM (nonmotile) that was isolated from a calf with diarrhea. zeta intimin showed the highest similarity (88%) of its amino acid sequence to the alpha intimin. For diagnostic purposes, we established a polymerase chain reaction (PCR) method for diagnosis of the key virulence traits of VTEC (i.e., verotoxins and intimins). This method also distinguishes between the toxins (VT1 and VT2) and the six intimin types. By applying the PCR method, intimin zeta in strains of other VTEC serotypes O84:H2, O92:NM, O119:H25, and O150:NM was identified. Because the intimin types represent distinctive phylogenetic E. coli lineages, application of the intimin subtyping PCR offers significant benefits. These include improving diagnosis of VTEC infection and increasing the understanding of evolution of attaching and effacing VTEC and other LEE-positive bacteria.     

Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques

AIMS: The aim of this study was to isolate Escherichia coli O26, O103, O111 and O145 from 745 samples of bovine faeces using (i) immunomagnetic separation (IMS) beads coated with antibodies to lipopolysaccharide, and slide agglutination (SA) tests and (ii) PCR and DNA probes for the detection of the Verocytotoxin (VT) genes. METHODS AND RESULTS: IMS-SA tests detected 132 isolates of presumptive E. coli O26, 112 (85%) were confirmed as serogroup O26 and 102 had the VT genes. One hundred and twenty-two strains of presumptive E. coli O103 were isolated by IMS-SA, 45 (37%) were confirmed as serogroup O103 but only one of these strains was identified as Verocytotoxin-producing E. coli (VTEC). Using the PCR/DNA probe method, 40 strains of VTEC O26 and three strains of VTEC O103 were isolated. IMS-SA identified 21 strains of presumptive E. coli O145, of which only four (19%) were confirmed as serogroup O145. VTEC of this serogroup was not detected by either IMS-SA or PCR/DNA probes. E. coli O111 was not isolated by either method. CONCLUSION: IMS beads were 2.5 times more sensitive than PCR/DNA probe methods for the detection of VTEC O26 in bovine faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: IMS-SA is a sensitive method for detecting specific E. coli serogroups. However, the specificity of this method would be enhanced by the introduction of selective media and the use of tube agglutination tests for confirmation of the preliminary SA results.     

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.     

Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform.     

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.