Vero cytotoxin-producing Escherichia coli, particularly serogroup O157, associated with human infections in England and Wales: 1992-4.

Investigations were performed by the Laboratory of Enteric Pathogens on Vero cytotoxin-producing Escherichia coli (VTEC) in England and Wales from 1992-4. Bacterial isolates, faeces and sera obtained from patients with diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome were examined. Using serotyping, Vero cytotoxin gene probing and serodiagnostic tests for E. coli O157, evidence of infection was detected in 543, 434 and 491 individuals in 1992, 1993 and 1994 respectively; VTEC of serogroup O157 were isolated from 470, 385 and 411 cases. The O157 VTEC strains belonged to at least 19 different phage types (PT) although 84% belonged to PT2, PT49, PT8, PT1 or PT4. Antibodies to E. coli O157 lipopolysaccharide were detected in 13% of the cases. The average annual rate of infection with O157 VTEC was 0.83/100000 and 12% of the 1458 individuals with evidence of infection with VTEC or E. coli O157 developed haemolytic uraemic syndrome. There were at least 18 general outbreaks and many family outbreaks.     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

Extended phage-typing scheme for Escherichia coli O157:H7

In Canada, the number of human isolates of verotoxigenic (VT + ve) Escherichia coli O157:H7 from diarrhoeal cases and haemolytic uraemic syndrome and haemorrhagic colitis has increased from 25 in 1982 to 2384 in 1989. A total of 3273 VT + ve E. coli O157:H7 strains (3255 strains isolated in Canada and 18 isolates from other countries) were phage typed. The phage typing scheme has been extended from 14 to 62 phage types. Of these, five types occurred exclusively in other countries (type 47 in Japan; and types 49, 50, 51 and 52 in the U.K.). Thirty-five different phage types were identified in Canada; only nine of these (1, 2, 4, 8, 14, 21, 23, 31 and 32), each accounted for more than 1% of the cases from human sources. The same nine types were the only ones observed among the isolates from non-human sources (meat and slaughter houses) suggesting a food-borne transmission in most of the human cases. Phage types 1 (30.5%); 4 (21%); 8 (13.5%); 31 (8.9%) and 14 (8%) were encountered in varying frequencies in most of the provinces; infrequently occurring phage types also showed regional variation. Thirteen different phage types were identified among 151 outbreaks representing 556 isolates of E. coli O157:H7. More than one phage type were encountered in 12 outbreaks whereas in 141 outbreaks, all strains in each, had the same phage type.     

Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe.

Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.     

Verotoxin producing Escherichia coli O 157 infections associated with the consumption of yoghurt.

Sixteen cases of verotoxin producing Escherichia coli (VTEC) O 157:H7 Phage Type 49 infection were identified in the North West of England from 1 September to 1 November 1991, eight of whom lived in or around the same large town. Eleven of the cases were aged 10 years or less, and five of the affected children developed haemolytic uraemic syndrome. A case control study demonstrated a strong association between VTEC O 157:H7 PT 49 infection and the consumption of a locally produced live yoghurt. This is the first time that an outbreak of VTEC O 157 infection has been linked to the consumption of yoghurt and this vehicle of infection should be considered when investigating such outbreaks in future.     

Phage-typing of Vero-cytotoxin (VT) producing Escherichia coli O157 isolated in the United Kingdom

Vero-cytotoxin (VT) producing Escherichia coli serogroup O157 have been isolated from patients with diarrhoea, haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). A phage-typing scheme developed in Canada has been used to type 155 VT+ E. coli O157 serogroup isolated from sporadic infections in the UK since 1983, and 48 strains from HC or HUS outbreaks. Twelve phage types were identified of which three, types 49, 51 and 52, have not been found in North America. All strains carried a 60 x 10(6) plasmid and most VT1+VT2+ strains also had a 5 x 10(6) plasmid coding for colicin D production. The majority of strains producing both VT1 and VT2 belonged to phage type 1, or the related types 4, 8 and 14. Most strains producing only VT2 belonged to types 2 or 49. Four outbreaks were included in the survey. Three had strains of a single phage type while strains from the fourth outbreak were more variable. The distribution of phage types throughout the UK showed no marked geographical variations.     

Milkborne general outbreaks of infectious intestinal disease,England and Wales, 1992-2000

From 1 January 1992 to 31 December 2000, 27 milkborne general outbreaks of infectious intestinal disease (IID) were reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC). These outbreaks represented a fraction (2%) of all outbreaks of foodborne origin (N = 1774) reported to CDSC, but were characterized by significant morbidity. Unpasteurized milk (52%) was the most commonly reported vehicle of infection in milkborne outbreaks, with milk sold as pasteurized accounting for the majority of the rest (37%). Salmonellas (37%), Vero cytotoxin-producing Escherichia coli (VTEC) O157 (33%) and campylobacters (26%) were the most commonly detected pathogens, and most outbreaks were linked to farms (67%). This report highlights the importance of VTEC O157 as a milkborne pathogen and the continued role of unpasteurized milk in human disease.     

Asymptomatic carriage of verocytotoxin-producing Escherichia coli O157 in farm workers in Northern Italy

Faecal samples from 350 farm workers on 276 dairy farms and 50 abattoir employees from seven different operations were examined for the presence of Verocytotoxin-producing Escherichia coli 0157 (VTEC O157) by an O157-specific enzyme-linked fluorescent assay followed by immunoconcentration. VTEC O157 was isolated from four (1.1%) of the farm workers. A second stool sample was obtained from the positive farm workers as well as from their household contacts. VTEC O157 was isolated from the wife of one of them. The strains from the same household shared the same Verocytotoxin genes profile, phage type and pulsed-field gel electrophoresis pattern. The VTEC O157-positive subjects had neither intestinal symptoms at the moment of sampling nor a history of bloody diarrhoea or renal failure. Our study seems to confirm the hypothesis that farm residents often develop immunity to VTEC O157 infection, possibly due to recurrent exposure to less virulent strains of VTEC.     

The prevalence and characterization of verotoxin-producing Escherichia coli isolated from cattle and pigs in an abattoir in Hong Kong

The aim of the study was to define the prevalence of verotoxin-producing Escherichia coli (VTEC) in cattle and pigs in a Hong Kong abattoir. Faecal and carcass samples collected from 986 cattle and 487 pigs from an abattoir were tested for verotoxin (VT) by PCR and cytotoxicity assays. VTEC was isolated from 415 and 1-8% of cattle faecal and carcass samples and from 2.1 and 0.2% of porcine faecal and carcass samples, respectively. Amongst 409 VTEC isolates from cattle, 9 were serotype O157:H7 and eaeA+. The most prevalent vt genotype among bovine VTEC was vtl+vt2 (73.8%) and in porcine VTEC was vt2e+ (30%). None of the porcine VTEC isolates and 9.3% of the bovine VTEC isolates was eaeA+. The non-O157 serogroup VTEC isolates carrying eaeA and EHEC-hlyA belonged to serogroups O172, O15, O84, O91, O110 and O121. The local dietary preference for pork or chicken (rather than beef), the low VTEC carriage in pigs, the rarity of additional virulence factors (caeA) in VTEC isolated from cattle may explain the apparently low incidence of human diarrhoeal disease associated with VTEC in Hong Kong hitherto. However, the presence of non-O157 VTEC strains carrying the eacA virulence marker in cattle highlights the fact that sole reliance on sorbitol-MacConkey agar for screening human VTEC isolates may underestimate the human disease burden. The changing dietary habits of the population in Hong Kong reinforce the need for continued vigilance.     

Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002

Escherichia coli O157:H7 causes 73,000 illnesses in the United States annually. We reviewed E. coli O157 outbreaks reported to Centers for Disease Control and Prevention (CDC) to better understand the epidemiology of E. coli O157. E. coli O157 outbreaks (>or=2 cases of E. coli O157 infection with a common epidemiologic exposure) reported to CDC from 1982 to 2002 were reviewed. In that period, 49 states reported 350 outbreaks, representing 8,598 cases, 1,493 (17%) hospitalizations, 354 (4%) hemolytic uremic syndrome cases, and 40 (0.5%) deaths. Transmission route for 183 (52%) was foodborne, 74 (21%) unknown, 50 (14%) person-to-person, 31 (9%) waterborne, 11 (3%) animal contact, and 1 (0.3%) laboratory-related. The food vehicle for 75 (41%) foodborne outbreaks was ground beef, and for 38 (21%) outbreaks, produce.     

Vero cytotoxin-producing Escherichia coli O157 outbreaks in England and Wales, 1995: phenotypic methods and genotypic subtyping.

Vero cytotoxin-producing Escherichia coli O157 belonging to four phage types (PTs) caused 11 outbreaks of infection in England and Wales in 1995. Outbreak strains of different PTs were distinguishable by DNA-based methods. Pulsed-field gel electrophoresis best discriminated among strains belonging to the same PT, distinguishing six of the seven PT2 outbreak strains and both PT49 outbreak strains.     

Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork, and chicken in southwestern Ontario

Samples of ground beef (225), pork (235) and chicken (200) were randomly selected from meat processing plants in the southwestern Ontario area. Supernatants of broth cultures of the samples were tested for verocytotoxins using a Vero cell assay. Neutralization of cytotoxic activity using antisera specific for three types of verocytotoxin (Verotoxin 1, Verotoxin 2 and Shiga-like toxin II) was performed on positive samples. Isolation of verocytotoxigenic Escherichia coli (VTEC) was attempted from positive samples. VTEC were confirmed as E. coli biochemically, tested for drug resistance, and serotyped. Based on neutralization studies, the prevalence of VTEC in beef and pork was at least 36.4% and 10.6%, respectively. This is much higher than has been reported from a survey of retail meats in which a method designed to detect only E. coli O 157.H7 was used. Isolations of VTEC were made from 10.4% of the beef samples and 3.8% of the pork samples. No VTEC were recovered from the chicken samples. The majority of VTEC isolates were susceptible to commonly used antimicrobial agents. A number of the serotypes of the VTEC isolates recovered have been associated with human disease; however, no VTEC of serotype O 157.H7 were isolated.     

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.     

Vero cytotoxin-producing Escherichia coli O157 gastroenteritis in farm visitors,North Wales

An outbreak of Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) gastroenteritis in visitors to an open farm in North Wales resulted in 17 primary and 7 secondary cases of illness. E. coli O157 Vero cytotoxin type 2, phage type 2 was isolated from 23 human cases and environmental animal fecal samples. A case-control study of 16 primary case-patients and 36 controls (all children) showed a significant association with attendance on the 2nd day of a festival, eating ice cream or cotton candy (candy floss), and contact with cows or goats. On multivariable analysis, only the association between illness and ice cream (odds ratio [OR]=11.99, 95% confidence interval [CI] 1.04 to 137.76) and cotton candy (OR=51.90, 95% CI 2.77 to 970.67) remained significant. In addition to supervised handwashing, we recommend that foods on open farms only be eaten in dedicated clean areas and that sticky foods be discouraged.     

The role of Escherichia coli O 157 infections in the classical (enteropathic) haemolytic uraemic syndrome: results of a Central European, multicentre study.

To assess the importance of infection by Verotoxin (VT) producing Escherichia coli (VTEC) in children with HUS in Central Europe, stool and/or serum samples obtained from 147 patients from 28 paediatric centres were prospectively examined for the presence of VTEC and the kinetics of faecal VT titres (FVT), and for VT neutralization titres and antibodies against E. coli O 157 lipopolysaccharide, respectively. Ninety-two percent of the patients had classic (enteropathic) HUS (E+ HUS). Evidence of VTEC infection was obtained in 86% of them. VTEC/FVT were identified in 55/118 E+ cases (47%). A prominent feature was the frequent isolation of sorbitol-fermenting, VT2-producing E. coli O 157.H-.VT1 (C600/H19) was neutralized by 9%, and VT2 (C600/933W) by 99% of the initial serum samples from E+ patients, compared to 3% (VT1) and 100% (VT2) from age-related controls. Fourfold titre rises against VT1 and/or VT2 were observed in 13/70 (19%), and significantly elevated O 157 LPS IgM and/or IgA antibodies in 106/128 (83%) of the E+ patients. The ubiquitous VT2 neutralizing principle in the serum of HUS patients as of healthy controls warrants further investigations.     

A foodborne outbreak of Vero cytotoxin-producing Escherichia coli O157:H-phage type 8 in hospital.

This paper describes the epidemiological and microbiological aspects of the largest outbreak of Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) infection in a hospital setting in which the route of transmission was foodborne. The outbreak, which was caused by a relatively uncommon phage type of VTEC O157, occurred in four geriatric continuing care wards in May 1997. The total number of people found to be excreting the organism was 37, of whom 16 were inpatients and 11 were staff. Twelve people displayed enteric symptoms. In addition, all but two of 10 cases identified in the local community were thought to be associated with the outbreak. An epidemiological investigation amongst the hospital patients revealed a statistically significant association between VTEC O157 infection and attendance at a concert party on the continuing care wards on 17 May 1997 (relative risk = 3.22;P= 0.006). There was an even stronger relationship between consumption of home-baked cream-filled cakes brought to that party and evidence of infection (relative risk = 19.35;P= 0.00002). Further investigations in the local community, coupled with microbiological evidence, supported the epidemiological finding that homemade cream cakes brought into the hospital were the vehicle of infection for the outbreak. There was no secondary spread within the hospital. The outbreak serves as a reminder of the hazard posed by foodstuffs brought into a hospital from outside.     

Genetic characterization of non-O157 verocytotoxigenic Escherichia coli isolated from raw beef products using multiple-locus variable-number tandem repeat analysis

Verocytotoxigenic Escherichia coli (VTEC) can produce serious human illness linked to the consumption of contaminated food, mainly of bovine origin. There is growing concern about non-O157 VTEC serotypes, which in some countries cause severe infections in a proportion similar to O157:H7 strains. As several epidemiological studies indicated the important role of meat as the major vehicle in the transmission of this pathogen to human consumers, our aim was to investigate the genetic diversity among non-O157:H7 VTEC isolated from raw beef products. We performed a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), and to our knowledge, this is the first time that VTEC serotypes O8:H19, O112:H2, O113:NM, O171:NM, ONT:H7, ONT:H19, and ONT:H21 were typed by this method. MLVA typing grouped the total number of strains from this study (51) into 21 distinct genotypes, and 11 of them were unique. Several MLVA profiles were found in different serotypes, O178:H19 being the most variable. The isolates could be principally discriminated by alleles of three of seven loci studied (CVN001, CVN004, and CVN014), and on the other hand, CVN003 rendered null alleles in all the isolates. As some VNTR markers might be serotype specific, it is possible that the implementation of new VNTR loci will increase intraserotype discrimination.     

Outbreak of Vero cytotoxin-producing Escherichia coli O157 in a child day care facility

In December 1998, an outbreak of Vero cytotoxin-producing Escherichia coli (VTEC) O157 in a crèche affected ten out of 45 children and one out of five staff members. Eight cases were symptomatic and three were asymptomatic. There were two asymptomatic adult family contacts of child cases. All specimens were identified as VTEC O157:H7, phage type 32. None of the cases were seriously ill and none developed haemolytic uraemic syndrome (HUS). One child continued to excrete the organism for 14 weeks. The origin of the outbreak was not found but epidemiological investigation was suggestive of person-to-person spread. All children and staff were screened and excluded from the crèche until microbiological clearance was obtained. An inspection of the crèche revealed overcrowding and inadequacies in cleaning and in the food preparation facilities. These problems were remedied before children were re-admitted to the crèche. This outbreak demonstrates the ease with which VTEC O157 can be transmitted between small children. Two specific features of this outbreak were notable: (1) the mild self-limiting nature of the illness and (2) the prolonged shedding of the bacterium by one child.     

Virulence profiling and disease association of verocytotoxin-producing Escherichia coli O157 and non-O157 isolates in Belgium

Whereas the association of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 with the hemolytic uremic syndrome (HUS) is well established, the medical importance of many non-O157 serotypes remains unclear. Using polymerase chain reaction (PCR), we have investigated the distribution of the pathogenicity island O island 122 (OI-122) and other virulence genes in VTEC belonging to seropathotypes (SPT) A through D, and assessed their association with human disease. Two hundred sixty-five VTEC isolated from human stools comprising 52 O157 (of which 14 associated with HUS) and 213 non-O157 isolates (of which 19 associated with HUS) were studied. A complete OI-122 (COI-122) was detected in all O157, but in only 35 (16.4%) of non-O157 strains. A progressive decrease in the frequency of COI-122 was observed from SPT A through D, with a concomitant increase in the frequencies of incomplete and absent OI-122. We focused on the variable virulence profiles of the non-O157 serotypes and found that COI-122 was also more frequently present in isolates associated with HUS (p=0.001). The individual genes vtx2, eae, espP, as well as the OI-122-associated genes sen, nleB, nleE, and the efa gene cluster were significantly more often present in non-O157 VTEC associated with HUS. Non-O157 isolates carrying the combined virulence profile vtx2-nleE-efa showed the strongest association with HUS (p<0.0001). Molecular risk assessment by determination of virulence profiles of individual isolates may be useful in the identification of highly virulent non-O157 strains. We showed that the detection of a specific gene combination could assist in identifying non-O157 VTEC isolates that pose a serious public health concern.     

Vero cytotoxin-producing Escherichia coli, particularly serogroup O 157, associated with human infections in the United Kingdom: 1989-91.

This survey reports the results of investigations performed by the Laboratory of Enteric Pathogens (LEP), to identify evidence of human infection with Vero cytotoxin-producing Escherichia coli (VTEC) in the UK during the period 1989-91. Bacterial isolates, faecal specimens and serum samples were received from patients suffering from diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome. Using serotyping, Vero cytotoxin gene probing and an ELISA for serum antibodies to E. coli O 157, evidence of infection was detected in 232, 428 and 615 individuals in 1989, 1990 and 1991 respectively. Of these individuals, 15% were reported as having HUS. Vero cytotoxin-producing E. coli O 157 was the most frequently encountered serogroup, with isolations from a total of 1092 individuals over the 3-year period. The incidence of VTEC infection increased from 0.41/100,000 in 1989 to 1.07/100,000 in 1991. The area with the highest rate of infection in each year was Scotland, increasing from 1.37/100,000 in 1989 to 3.97/100,000 in 1991.