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.     

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.     

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.     

Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

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.     

COMPARISON OF RAPID TECHNIQUES FOR THE DETECTION OF ESCHERICHIA COLI O157:H7

Four different commercial kits (EHEC-TEK of Organon Teknika, Durham, NC; HEC O157 of 3M, St. Paul, MN; and Coline dipstick and Coline one-step of AMP-COR Inc., Camden, NJ) were evaluated for the detection and recovery of E. coli O157:H7from 75 fresh meat samples and 23 artificially inoculated beef and pork samples. Of the total 75 samples tested, 21 (28%) were presumptive positive by HEC O157 and Coline dipstick, 18 (24%) by Coline one-step, and 12 (16%) by EHEC-TEK. None of the presumptive positive samples by any of the methods was confirmed as E. coli O157:H7 (false positive). Of the 23 positive spiked samples tested, 23 were recovered by Coline dipstick and one-step (100%), 22 (95.6%) by HEC O157, and 20 (86.9) were recovered by EHEC-TEK. In the confirmation step 17 of the 23 spiked samples produced characteristic colonies on MacConkey sorbitol agar (Difco) with 5-bromo-4-chloro-3-indoxy-β-D-glucuronide (Biosynth International) (MSA-BCIG) and were confirmed as E. coli O157:H7. No characteristic colonies on MSA-BCIG were detected for 6 of the spiked samples with initial inoculum levels of between 2 to 70 cells/g and, therefore, were not confirmed as E. coli O157:H7. A better enrichment medium, as well as improved selective plating and confirmation techniques, are needed to enhance the selective growth of E. coli O157:H7 and provide lower detection levels.     

An eight-month study of a population of verocytotoxigenic Escherichia coli (VTEC) in a Scottish cattle herd

AIMS: Strains of Verocytotoxin-producing Escherichia coli (VTEC) from Scottish beef cattle on the same farm were isolated during four visits over a period of eight months. Characteristics of these strains were examined to allow comparisons with strains of VTEC associated with human infection. METHODS AND RESULTS: Strains were characterized to investigate the relationship between these bovine isolates with respect to serotype, Verocytotoxin (VT) type, intimin-type, and presence or absence of the enterohaemolysin genes. VT genes were detected in 176 of 710 (25%) faecal samples tested using PCR, although only 94 (13%) VTEC strains were isolated using DNA probes on cultures. Forty-five different serotypes were detected. Commonly isolated serotypes included O128ab:H8, O26:H11 and O113:H21. VTEC O26:H11 and O113:H21 have been associated with human disease. Strains harbouring the VT2 genes were most frequently isolated during the first three visits to the farm and those with both VT1 and VT2 genes were the major type during the final visit. Of the 94 strains of non-O157 VTEC isolated, 16 (17%) had the intimin gene; nine had the gene encoding beta-intimin and seven strains had an eta/zeta-intimin gene. Forty-one (44%) of 94 strains carried enterohaemolysin genes. CONCLUSIONS: Different serotypes and certain transmissible characteristics, such as VT-type and the enterohaemolysin phenotype, appeared to be common throughout the VTEC population at different times. SIGNIFICANCE AND IMPACT OF THE STUDY: Detailed typing and subtyping strains of VTEC as described in this study may improve our understanding of the relationship between bovine VTEC and those found in the human population.     

Non-O157 verotoxin-producing Escherichia coli: a problem,paradox, and paradigm

The problems associated with identification and characterization of non-O157 verotoxin-producing Escherichia coli (VTEC) are discussed. The paradox of VTEC is that most reports of human illnesses are associated with serotypes such as O157:H7, O111:H- (nonmotile), O26:H11, and O113:H21, which are rarely found in domestic animals. However, those VTEC serotypes commonly found in domestic animals, especially ruminants, rarely cause human illnesses. When they cause human illnesses, the symptoms are similar to those caused by the serotypes E. coli O157:H7, O111:H-, O26:H11, and O113:H21. The impact of VTEC on human and animal health is also addressed. The VTEC and their toxicity are considered as a paradigm for emerging pathogens. The question on how such pathogens could arise from a basic commensal population is also addressed.     

Detection of Escherichia coli O157 in French food samples using an immunomagnetic separation method and the VIDASTME. coli O157

C. VERNOZY-ROZAND, C. MAZUY, S. RAY-GUENIOT, S. BOUTRAND-LOEï, A. MEYRAND AND y. richard. 1997. Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS^TM E. coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples. A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined. Four poultry, one raw sausage and one ground beef sample were found to be positive for E. coli O157 by both methods. Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E. coli .     

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.     

Prevalence and characteristics of verotoxin-producing Escherichia coli (VTEC) in stool samples from asymptomatic human carriers working in the meat processing industry in Switzerland

A total of 5590 stool samples from healthy employees in the meat industry were screened by PCR for verotoxin-producing Escherichia coli (VTEC). The PCR product of VT-encoding genes was detected in 3. 5% of the samples. Phenotypic and genotypic traits of 47 VTEC strains isolated from asymptomatic carriers were characterized. A variety of serotypes was found; one strain belonged to the serotype O157:H7. The majority of the isolates proved to be VT2-positive. Fifty-seven percent of the verotoxin-producing strains harboured the genes for one or several additional virulence associated factors, including intimin (eae, 8.5%), the 60 MDa plasmid (42.5%), enterohaemolysin (EHEC-hlyA, 38.3%), the heat-stable enterotoxin (astA, 6.4%), a serin protease (espP, 6.4%), colicin production (col D157, 12.8%) and a secretion system II (etpD, 10.6%). None of the strains was positive for a specific enzyme with catalase-peroxidase activity (katP).     

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.     

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.     

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.     

Serotypes and antibiotic resistance of Verotoxigenic (VTEC) and Necrotizing (NTEC) Escherichia coli strains isolated from calves with diarrhoea

Serotypes and antibiotic resistance of 51 Verotoxigenic (VTEC) and 33 Necrotizing (NTEC) bovine Escherichia coli strains were determined and compared with those shown by 205 non-VTEC non-NTEC strains isolated from the same batch of calves. E. coli untypable for O-antigen represented 47% of the VTEC, 12% of the NTEC and 8.8% of the non-VTEC non-NTEC. Typable VTEC belonged to serotypes 02:K?, 0103:K-, 0104:K?, 0128:K?, 0153:K- and O157:K-:H7, whereas typable NTEC were of serotypes 08:K87, 015:K14, 015:K-, 054:K?, 076:K-, 078:K(80), 088:K?, 0123:K-, 0139:K- and 0153:K-. Non-VTEC non-NTEC showed a wide variety of serotypes which were generally unrelated to those found in VTEC and NTEC. VTEC were resistant to antibiotics at higher rates than NTEC and non-VTEC non-NTEC, and showed also the highest multidrug-resistant pattern. Our results show that bovine VTEC strains belonged to O-groups usually found in human VTEC causing sporadic diarrhoea, haemorrhagic colitis and/or haemolytic uraemic syndrome, such as 02, 0103, 0104, 0153 and especially 0128 and O157. In contrast, bovine NTEC strains belonged to serotypes different from those previously found in necrotizing E. coli strains of human origin.     

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.     

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.     

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.