The serological response to Verocytotoxigenic Escherichia coli in patients with haemolytic uraemic syndrome

AIMS: To screen sera from 80 patients with clinical haemolytic uraemic syndrome (HUS) and serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157, for antibodies to Verocytotoxin-producing Escherichia coli (VTEC) belonging to serogroups O5, O26, O104, O111, O128, O145, O153 and O165. METHODS AND RESULTS: Sera were screened by an LPS-based ELISA and SDS-PAGE/immunoblotting. None of the 80 sera contained antibodies binding to long-chain LPS of any of the LPS types employed; however, nine sera contained antibodies binding to R3 LPS-core epitopes. CONCLUSIONS: The presence of patients' serum antibodies to the LPS of E. coli O157, in the absence of antibodies to the LPS of a range of other VTEC, demonstrated that cases of HUS may be caused by strains of O157 VTEC alone and that concurrent infection with multiple strains of VTEC is not a prerequisite for cases of HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibodies to long-chain LPS of VTEC other than O157 were not detected, and so there was no evidence of infection with VTEC belonging to more than one serogroup. The results of immunoassays such as ELISAs and micro-agglutinations must take into consideration antibodies binding to R3 epitopes located on LPS-core.     

Escherichia coli serogroup O26--a new look at an old adversary

Escherichia coli serogroup O26 played an important part in the early work on Verocytotoxin and is an established diarrhoeal pathogen. Recently, Verocytotoxigenic E. coli (VTEC) O26 has been increasingly associated with diarrhoeal disease and frequently linked to outbreaks and cases of haemolytic uraemic syndrome (HUS). This review investigates the pathogenicity, geographical distribution, changing epidemiology, routes of transmission and improved detection of VTEC O26. Laboratory data on VTEC O26 isolates and clinical data on HUS suggest a true difference in the incidence of VTEC O26 in different geographic locations. However, few diagnostic laboratories use molecular methods to detect VTEC and so it is difficult to assess the role of VTEC O26 in causing diarrhoeal disease. VTEC O26 is frequently found in the cattle population but rarely in food. However, the small number of outbreaks analysed to date are thought to be food-borne rather than associated with direct or indirect contact with livestock or their faeces. The increase in awareness of VTEC O26 in the clinical and veterinary setting has coincided with the development of novel techniques that have improved our ability to detect and characterize this pathogen.     

Human antibody responses to R3-core epitopes on the lipopolysaccharide of Escherichia coli O157

AIMS: To establish the incidence of serum antibodies binding to the R3-core lipopolysaccharide (LPS) of verocytotoxin-producing Escherichia coli (VTEC) O157, in patients with serum antibodies to E. coli O157 LPS, and to characterize the class(es) of antibodies binding to epitopes on the R3-core. METHODS AND RESULTS: SDS-PAGE profiles of LPS prepared from VTEC O157 were used in combination with immunoblotting to detect and characterize serum antibodies binding to the R3-core LPS of VTEC O157. Of 417 sera, referred to the Laboratory of Enteric Pathogens (LEP) for routine O157 serology and found to have serum antibodies to long-chain VTEC O157 LPS, 31 had antibodies binding to the R3-core of VTEC O157 LPS. The majority of the 31 sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. Patients who did not develop haemolytic uraemic syndrome (HUS) produced antibodies of the IgM class to R3-core and IgG-class antibodies to long-chain LPS more frequently than patients with HUS. CONCLUSIONS: Only 7.4% of sera received by the LEP, and shown to have antibodies to VTEC O157 LPS, contained antibodies binding to the R3-core of VTEC LPS. Most sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. SIGNIFICANCE AND IMPACT OF THE STUDY: Patients infected with VTEC O157 produced antibodies binding to the R3-core epitopes of VTEC O157 LPS only rarely, and these antibodies are unlikely to interfere with the serodiagnosis of infections caused by these organisms.     

Introduction to the food safety concerns of verotoxin-producing Escherichia coli

Verotoxin-producing Escherichia coli (VTEC) have emerged in the past two decades as food-borne pathogens that can cause major outbreaks of human illnesses worldwide. The number of outbreaks has increased in recent years due to changes in food production and processing systems, eating habits, microbial adaptation, and methods of VTEC transmission. The human illnesses range from mild diarrhea to hemolytic uremic syndrome (HUS) that can lead to death. The VTEC outbreaks have been attributed to O157:H7 and non-O157:H7 serotypes of E. coli. These E. coli serotypes include motile (e.g., O26:H11 and O104:H21) and nonmotile (e.g., O111:H-, O145:H-, and O157:H-) strains. In the United States, E. coli O157:H7 has been the major cause of VTEC outbreaks. Worldwide, however, non-O157:H7 VTEC (e.g., members of the O26, O103, O111, O118, O145, and O166 serogroups) have caused approximately 30% of the HUS cases in the past decade. Because large numbers of the VTEC outbreaks have been attributed to consumption of ruminant products (e.g., ground beef), cattle and sheep are considered reservoirs of these food-borne pathogens. Because of the food safety concern of VTEC, a global perspective on this problem is addressed (Exp Biol Med Vol. 228, No. 4). The first objective was to evaluate the known non-O157:H7 VTEC strains and the limitations associated with their detection and characterization. The second objective was to identify the VTEC serotypes associated with outbreaks of human illnesses and to provide critical evaluation of their virulence. The third objective was to determine the rumen effect on survival of E. coli O157:H7 as a VTEC model. The fourth objective was to explore the role of intimins in promoting attaching and effacing lesions in humans. Finally, the ability of VTEC to cause persistent infections in cattle was evaluated.     

Fenotypic and genotypic characterization of Shiga toxin-producing Escherichia coli O111 strain isolated from patient with hemolytic-uremic syndrome

Shiga toxin-producing E. coli O157 and non-O157 are important emergance pathogens that can cause diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome (HUS). A few cases of EHEC infections are documented per year in Poland. Among them only one patient with EHEC O157 infection developed HUS. We characterized the first VTEC non-O157 strain isolated from child with HUS in Poland. The VTEC O111 strain produced Stx2 which was cytotoxic for Vero cell. Using DNA microarray analysis we have found set of virulence genes in VTEC O111 strain as: stx2A, stx2B, ehly, eae, tir tccP espA, espJ, cif nleA, nleB, lpfA, iha, efa1, cba. The strain was fenotypic resistant to streptomycin, tetracyclin and sulphonamides (strA, tetA, sul2 genes were detected).     

The serodiagnosis of infections caused by Verocytotoxin-producing Escherichia coli

Patients with haemolytic uraemic syndrome (HUS) and haemorrhagic colitis (HC) produce serum antibodies to the lipopolysaccharides (LPS) of Escherichia coli O157 and certain other E. coli serogroups. Patients may also make salivary antibodies to the LPS of E. coli O157. Serological tests based on these antibodies can be used to provide evidence of infection in the absence of culturable VTEC or the toxins they produce. Serum antibodies to LPS persist for several months following onset of disease, enabling both current and retrospective serological testing. The LPS of E. coli O157 shares epitopes with strains of Brucella abortus, Yersinia enterocolitica O9, Vibrio cholerae O1 Inaba, group N Salmonella and certain strains of Citrobacter freundii and E. hermanni. Serological tests for serum antibodies to E. coli O157 should be evaluated in the light of these cross-reactions. Serological tests to supply evidence of infection with E. coli O157 have been shown to provide a valuable adjunct to bacteriological procedures for detecting culturable VTEC and VT. The use of well characterized LPS antigens in association with the techniques of ELISA and immunoblotting provide valuable procedures for detecting evidence of infection with E. coli O157 and possibly other VTEC.     

Inhibitory effect of creosote and its main components on production of verotoxin of enterohaemorrhagic Escherichia coli O157:H7

The inhibitory effects of creosote and its main components, alpha-methoxyphenol and o-ethylphenol, on production of verotoxin by enterohaemorrhagic Escherichia coli O157 (VTEC E. coli) were investigated. The production of verotoxin by VTEC E. coli was inhibited by the administration of 0.001-0.1% of creosote or o-ethylphenol (final concentration). On the other hand, weak inhibition of production of verotoxin was observed with 0.1% alpha-methoxyphenol administration. As the inhibitory effects were obtained below Minimal Inhibitory Concentration (MIC) values, these test compounds exerted their effects at the active site of VTEC E. coli cells prior to their production of verotoxin. These findings suggest that pre-administration of creosote and its main components might prevent human intestinal food poisoning by VTEC E. coli and contribute to maintenance of public health.     

Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E. coli toxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.     

Verocytotoxin-producing Escherichia coli: a veterinary view

This overview places verocytotoxin-producing Escherichia coli (VTEC) in perspective with other E. coli types that cause disease in animals. VTEC O157 and other verocytotoxin-producing serotypes cause severe disease in man but to date, although other VTEC are found in animals, zoonosis appears to be associated with E. coli O157 only. The epidemiology of E. coli O157 in cattle has been studied in Scotland, and this work is described alongside current knowledge.     

Verocytotoxin-producing Escherichia coli in wild birds and rodents in close proximity to farms

Wild animals living close to cattle and pig farms (four each) were examined for verocytotoxin-producing Escherichia coli (VTEC; also known as Shiga toxin-producing E. coli). The prevalence of VTEC among the 260 samples from wild animals was generally low. However, VTEC isolates from a starling (Sturnus vulgaris) and a Norway rat (Rattus norvegicus) were identical to cattle isolates from the corresponding farms with respect to serotype, virulence profile, and pulsed-field gel electrophoresis type. This study shows that wild birds and rodents may become infected from farm animals or vice versa, suggesting a possible role in VTEC transmission.     

Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.     

Virulence factors of verocytotoxin-producing Escherichia coli isolated from raw meats.

PCR for verocytotoxin-producing Escherichia coli (VTEC) was positive in 4.6% of 2,440 raw meat samples; only beef, sheep, and venison samples were positive. None of the isolated VTEC strains belonged to serogroup O157. Additional virulence factors were detected in only a minority of strains, suggesting that most of these meat VTEC isolates are not pathogenic.     

Verotoxin-producing Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) isolated from healthy cattle in Spain

AIMS: To determine the prevalence and characteristics of verotoxigenic Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) in healthy cattle. METHODS AND RESULTS: Faecal samples from 412 healthy cattle were screened for the presence of VTEC, EPEC and NTEC. Four isolates from each sample were studied. VTEC, EPEC and NTEC were isolated in 8.7%, 8.2% and 9.9% of the animals, respectively. VTEC and NTEC were isolated more frequently from calves and heifers than from adults. Seventy (4.2%), 69 (4.2%) and 74 (4.5%) of the 1648 E. coli isolates were VTEC, EPEC and NTEC, respectively. Seventeen (24.3%) of the VTEC strains were eae-positive. Thirty-six (51.4%) of VTEC strains belonged to E. coli serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. The serogroups most prevalent among the EPEC strains were O10, O26, O71, O145 and O156. CONCLUSIONS: Healthy cattle are a reservoir of VTEC, EPEC and NTEC. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most of the VTEC strains were eae-negative, a high percentage of VTEC strains belonged to serogroups associated with severe disease in humans.     

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.     

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.     

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.     

Identification of enterohaemorrhagic Escherichia coli by means of their production of enterohaemolysin

With increasing interest in verocytotoxigenic Escherichia coli (VTEC) being associated with both human and animal infection, a simple system of rapidly identifying the majority of VTEC has been devised. This depends on the fact that most VTEC produce enterohaemolysin, which is rarely produced by non-VTEC. By employing two media, traditional sheep blood agar (SBA) and washed sheep blood agar supplemented with calcium (WSBA-Ca), enterohaemolysin-producing strains can be easily differentiated from other E. coli enabling most VTEC types, including ones belonging to serogroups other than O157, to be isolated.     

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.     

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.     

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.