The aim of this study was to characterise the atypical enteropathogenic Escherichia coli (EPEC) strains isolated during a study of intestinal infectious disease in the UK by serotyping, intimin subtyping, and antimicrobial resistance typing. Serotypes, intimin subtypes, and resistance patterns of strains from cases were then compared with those from the control group. A wide range of serotypes, intimin subtypes, and antimicrobial resistance patterns was identified in isolates from both cases and controls, with O70:H11 and O111:H- being the most frequently detected serotypes. The most common intimin types were gamma and gamma(2). Thirty-six percent of the EPEC isolates were resistant to at least one antimicrobial agent. No significant differences in the characteristics of EPEC strains isolated from patients with symptoms of gastrointestinal disease versus those isolated from healthy controls were detected, although strains harbouring the beta-intimin subtype were more commonly isolated from children under 5 years of age (p=0.002). The compilation of data on atypical EPEC strains presented here indicates the need for further study of their virulence and epidemiology in order to assess their significance as human pathogens.

Foodborne outbreaks of Salmonella enterica serovar Enteritidis phage type 4 (PT4) infection (n=497), reported to the Health Protection Agency Communicable Disease Surveillance Centre between 1992 and 2002, were compared with other pathogens (n=1148) to determine factors (season, setting, food vehicles, food safety faults) associated with this pathogen. Logistic regression was applied to control for potential confounding. Foodborne general outbreaks of S. Enteritidis PT4 infection were more likely to occur in the spring and summer, and were more often linked to schools, private residences and residential institutions. Eggs, egg products and the use of raw shell egg were strongly associated with this pathogen. Most outbreaks were linked to cross-contamination and inadequate heat treatment. This paper describes the decline in the S. Enteritidis PT4 epidemic, providing evidence that control measures introduced, e.g. improved biosecurity and vaccination, have worked. Continued surveillance of human and veterinary salmonellosis is essential to detect future problems.

A DNA microarray was used to analyze the distribution of plasmid and chromosomal genes among strains of enteroaggregative Escherichia coli (EAEC) isolated from a prospective diarrhoea surveillance study in the United Kingdom. Target genes were extracted from existing databases and from the genome sequence of prototype EAEC strain 042. We found that strains exhibiting the aggregative adherence (AA) phenotype could be broadly divided into two groups depending upon whether they harboured genes from the EAEC virulence plasmid (pAA) and a set of chromosomal genes found in EAEC strain 042. Several chromosomal loci were inherited en bloc, and were more common in strains which we designated Group 1; genes at the pheU locus were particularly conserved. Genes encoded on the pAA plasmid and those under control of the master regulator AggR were also concentrated in the Group 1 EAEC. A gene encoding a type 1 pilin allele was detected more frequently in Group 2 EAEC. Our data suggest that strains previously designated as typical EAEC harbour a large number of conserved plasmid and chromosomal loci, further illuminating a package of virulence genes common to the most important EAEC.

We conducted prospective surveillance of childhood hemolytic uremic syndrome (HUS) from 1997 to 2001 to describe disease incidence and clinical, epidemiologic and microbiologic characteristics. We compared our findings, where possible, with those of a previous study conducted from 1985 to 1988. The average annual incidence of HUS for the United Kingdom and Ireland (0.71/100,000) was unchanged from 1985 to 1988. The overall early mortality had halved, but the reduction in mortality was almost entirely accounted for by improved outcome in patients with diarrhea-associated HUS. The principal infective cause of diarrhea-associated HUS was Shiga toxin-producing Escherichia coli O157 (STEC O157), although in the 1997-2001 survey STEC O157 phage type (PT) 21/28 had replaced STEC O157 PT2 as the predominant PT. The risk of developing diarrhea-associated HUS was significantly higher in children infected with STEC O157 PT 2 and PT 21/28 compared with other PTs. Hypertension as a complication of HUS was greatly reduced in patients with diarrhea-associated HUS.

We investigate the epidemiology of 12 Verocytotoxin-producing Escherichia coli (VTEC) serogroups observed in a calf cohort on a Scottish beef farm. Fitting mathematical models to the observed time-course of infections reveals that there is significant calf-to-calf transmission of VTEC. Our models suggest that 40% of all detected infections are from calf-to-calf transmission and 60% from other sources. Variation in the rates at which infected animals recover from infection by different VTEC serogroups appears to be important. Two thirds of the observed VTEC serogroups are lost from infected calves within 1 day of infection, while the rest persist for more than 3 days. Our study has demonstrated that VTEC are transmissible between calves and are typically lost from infected animals in less than 1 week. We suggest that future field studies may wish to adopt a tighter sampling frame in order to detect all circulating VTEC serogroups in similar animal populations.

Two temporary Escherichia coli O group strains OX3 and OX7 are given permanent status as O174 and O175, respectively. Both these test strains were originally isolated from cases of human diarrhoea. Whereas the O174 strain is negative for known virulence genes, the O175 strain is positive with the probe derived from the CVD432 plasmid associated with the aggregative adherence phenotype, the Enteroaggregative heat-stable enterotoxin 1 gene (astA) and daaC (F1845 afimbrial adhesin) associated with the diffuse adherence (DA) phenotype. Additionally, six E. coli strains are established as antigenic test strains for six new O groups, designated O176, O177, O178, O179, O180 and O181. All six strains produced Verocytotoxin and were positive for vtx1, vtx2, or both genes. Additional virulence genes associated with diarrhoeal disease in humans were found in four of the strains. O176 and O177 strains were isolated from calves, O178 and O181 strains from meat, the O179 strain was from human bloody diarrhoea, and the O180 strain from swine. Preliminary data on the occurrence and epidemiology of these eight new O groups amongst groups of diarrhoeagenic E. coli are reviewed.

This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx(1), eae, and ehl genes, 6.5% carried vtx(1) and vtx(2), and one isolate carried vtx(2) only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx(2), and none carried vtx(1). Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx(1) or vtx(2). All but one serogroup O157 isolate carried vtx(2), eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.

ABSTRACTRectal fecal samples were taken once a week from 49 calves on the same farm. In addition, the dams of the calves were sampled at the time of calf birth and at the end of the study. Strains of verocytotoxin-producingEscherichia coli(VTEC) were isolated from these samples by using PCR and DNA probe hybridization tests and were characterized with respect to serotype, verocytotoxin gene (vtx) type, and the presence of the intimin (eae) and hemolysin (ehxA) genes. A total of 170 VTEC strains were isolated during 21 weeks from 130 (20%) of 664 samples from calves and from 40 (47%) of 86 samples from their dams. The characteristics of the calf strains differed from those strains isolated from the dams with respect to verocytotoxin 2 and the presence of theeaegene. In addition, no calf shed the same VTEC serogroup (excluding O?) as its dam at birth or at the end of the study. The most frequently detected serogroups in calves were serogroup O26 and provisional serogroup E40874 (VTEC O26 was found in 25 calves), whereas in dams serogroup O91 and provisional serogroup E54071 were the most common serogroups. VTEC O26 shedding appeared to be associated with very young calves and declined as the calves aged, whereas VTEC O2 shedding was associated with housing of the animals. VTEC O26 strains from calves were characterized by the presence of thevtx1,eae, andehxAgenes, whereasvtx2was associated with VTEC O2 and provisional serogroup E40874. The high prevalence of VTEC O26 and of VTEC strains harboring theeaegene in this calf cohort is notable because of the association of the O26 serogroup and the presence of theeaegene with human disease. No association between calf diarrhea and any of the VTEC serogroups was identified.

ABSTRACT Certain strains of Shiga toxin-producing Escherichia coli (STEC) which do not have the locus of enterocyte effacement pathogenicity island carry the STEC autoagglutinating adhesin ( saa ) gene. The distribution of the saa gene in STEC isolates from patients with hemolytic-uremic syndrome (HUS), patients with less severe diarrheal disease, asymptomatic individuals, and healthy cattle was examined. saa -positive strains were detected more frequently ( P < 0.001) in STEC strains from bovines (32 of 56 strains) than in those from humans (8 of 91 strains). No significant association ( P = 0.135) was found between the saa gene and STEC isolated from patients with HUS (6 of 46 strains) or diarrhea (2 of 29 strains) and from healthy controls (0 of 16 strains).

Verocytotoxin-producing Escherichia coli (VTEC) causes a wide spectrum of disease in humans, from mild diarrhoea to haemolytic uraemic syndrome (HUS). The verocytotoxin (vtx) and intimin (eae) genes of VTEC strains, other than those of serogroup O157, were subtyped to identify common properties that may be associated with increased pathogenicity. Strains were isolated from patients with HUS, those with diarrhoea or from asymptomatic individuals. Strains of VTEC that carried vtx(2) gene subtypes vtx(2) and vtx(2c) were most commonly associated with HUS, whereas strains from patients with less severe disease and from the healthy control group were more likely to have vtx(1c) or vtx(2d) genes. The eae gene was detected more frequently in strains isolated from HUS patients than in those associated with cases of diarrhoea; beta-intimin was the most common intimin subtype in strains isolated from both groups of patients. None of the strains from the healthy control group carried the eae gene.

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.

PCR-RFLP methods for subtyping the intimin gene from strains of typical and atypical enteropathogenic Escherichia coli (EPEC) and Verocytotoxin-producing E. coli (VTEC) were compared. A novel HhaI PCR-RFLP method was developed that was rapid, easy to use and amplified an 1852 bp fragment of the intimin gene from all isolates examined. This method was used to investigate the intimin sub-types of EPEC strains associated with 14 outbreaks of diarrhoeal disease between 1967 and 2001, and 20 sporadic cases between January and December 2000, in the UK and Eire. In this study, genes encoding α, β, γ, δ and ζ-intimin were detected in the EPEC strains associated with outbreaks and β, γ, ϵ, θ and ζ-intimin genes were identified in isolates from sporadic cases. The β-intimin gene was the most frequently detected sub-type in both the outbreak and sporadic strains.

From July through September 2000, patients in five European countries were infected with a multidrug-resistant strain of Salmonella Typhimurium DT204b. Epidemiologic investigations were facilitated by the transmission of electronic images (Tagged Image Files) of pulsed-field gel electrophoresis profiles. This investigation highlights the importance of standardized protocols for molecular typing in international outbreaks of foodborne disease.

An investigation of infectious intestinal disease in England included examination of feces for Vero cytotoxin-producing Escherichia coli (VTEC). Using DNA probe hybridization 27 VTEC strains were identified, 12 were from cases, and of these three belonged to serogroup O157. The remaining 15 strains were isolated from controls. The strains were confirmed biochemically as E. coli, they were serotyped and characterized according to their toxin production, the presence of sequences encoding intimin (eae) and enterohemolysin was determined and resistance to antimicrobial agents was determined. Six of the nine cases with non-O157 VTEC were less than 16 years old, only two of the 15 controls were under 16. Infection with more than one micro-organism was also considered.

In June 2001, as part of a microbiological study of bagged, ready-to-eat salad products, Salmonella enterica serotype Newport was isolated from a sample of pre-packed green salad distributed by a major supermarket retailer. The strain was characterised by phage typing, plasmid profile typing and pulsed-field gel electrophoresis. Other isolates of S. Newport from cases of human infection in England and Wales in the first six months of 2001 were similarly characterised. Of 60 strains from cases of human infection, 19 were found to be indistinguishable from that isolated from the salad product. This study highlights the benefits of an integrated approach to outbreak investigations, involving the various elements of the PHLS and the Food Standards Agency, and acknowledges the full co-operation of the retailer in ensuring the rapid withdrawal of the contaminated product.

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.

A marked increase in sporadic cases of enteritis due to enterohemorrhagic Escherichia coli serogroup O157 occurred in Osaka City, Japan, during 1996. To elucidate why the number of cases had increased, the isolates were classified using phage typing, random amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis (PFGE). Fifty-seven percent of the isolates (105/184) belonged to the same phage type (PT-32) and gave the same PFGE pattern; the clone had been isolated during a 3-week period, with a peak on July 15. It was concluded that the majority of the cases identified in July 1996 formed an outbreak, although epidemiological links to a possible common source were not established. The possibility that this outbreak was part of a huge regional outbreak including children at primary schools in Sakai City was discussed.

Transmission of verocytotoxin (VT)-producing Escherichia coli (VTEC) occurs by three main routes. These comprise food- or water-borne infections, acquisition of disease by direct or indirect contact with animals and person-to-person spread. Phenotypic typing of VTEC belonging to serogroup O157 is achieved by phage typing and identification of VT type. These properties quickly provide evidence for the linkage of human cases and their association to potential sources. DNA-based subtyping methods such as pulsed field gel electrophoresis (PFGE) are generally required to increase discrimination of VTEC O157 strains so that the spread of specific strains can be monitored. Phenotypic and DNA-based methods were used in the investigation of 85 general outbreaks of VTEC O157 infection between 1995 and 1999. Results were used in conjunction with epidemiological data to provide direct or indirect evidence for the likely route of transmission. Detailed strain fingerprinting identified specific food vehicles and reservoirs of infection in animals. Typing supported the implementation of measures to control the spread of infection that included pasteurisation orders, product withdrawal, temporary closure of retail premises and open farms and the introduction of HACCP-based working practices. In outbreaks involving widely distributed foods, DNA-based examination of apparently sporadic isolates with the same phage and VT type as outbreak strains was performed to identify additional potential outbreak cases and estimate the spread of infection. Strain typing was applied in outbreaks in nurseries and other institutions to monitor person-to-person spread, including careers and their families and to assess the involvement of community cases occurring at the same time. Rapid exchange of epidemiological, microbiological and typing data will be increasingly important in investigation of VTEC O157 outbreaks.

A total of 3429 isolations of verocytotoxin-producing Escherichia coli O157 (VTEC O157) was confirmed from human sources in England and Wales during the period 1995-1998. The largest annual total was 1087 in 1997. Most infections occurred in the third quarter of each year. The overall rate of infection ranged from 1.28 to 2.10/100,000 population and showed regional variation. The highest incidence was in children aged 1-4 years. Annually, between 5% and 11% of strains were from patients who had travelled abroad. There were 67 general outbreaks of infection represented by 407 (11.9%) VTEC O157 isolates. Outbreaks involved transmission by contaminated food or water, person-to-person spread and direct or indirect animal contact, and five were associated with foreign travel. The majority (76%) of strains carried verocytotoxin (VT) 2 genes and 23.3% were VT1+VT2. Most strains had the flagellar antigen H7, but c. 14% were non-motile. Approximately 20% of isolates were resistant to antimicrobial agents, predominantly streptomycin, sulphonamides and tetracycline. In addition to VTEC O157, strains of serogroup O157 that did not possess VT genes were identified. These were either derivatives of VTEC O157 that had lost VT genes or were strains with H antigens other than H7 that have never been associated with VT production. Strains of VTEC other than O157 were characterised. Most were associated with diarrhoea, bloody diarrhoea or haemolytic uraemic syndrome and had virulence markers in addition to VT.

Strains of Escherichia coli, hybridising with a DNA probe for enteroaggregative E. coli (EAggEC), were isolated from patients with infectious intestinal disease (IID) or gastro-enteritis, and healthy controls during the study of IID in England. Of 3506 cases presenting with an IID, 160 (4.6%) had faecal EAgg-EC as compared with 46 (1.7%) of 2772 healthy controls, 53% of EAggEC isolated from each of the ‘case’ and the ‘control’ groups adhered in a ‘stacked-brick’ formation. Strains from cases and controls belonged to over 39 and 14 different serogroups respectively, and approximately half of the strains isolated did not react with antisera in the current somatic antigen serotyping scheme. Forty-nine cases with EAggEC (31%) had a known history of foreign travel. Over 50% of strains isolated from cases and controls were resistant to one or more of eight antimicrobials, and antimicrobial resistance was not statistically significantly more common among cases with a known history of foreign travel (p = 0.57). These data form part of the largest investigation carried out on these organisms in the UK to date and provide the most comprehensive analysis of strains of EAggEC isolated from the general population of England.