Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak.

AIMS: Evaluation of multilocus variable number tandem repeat analysis (MLVA) to subtype all isolates of Vero cytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales. METHODS AND RESULTS: Over a 13 month period from December 2010, 483 isolates of VTEC O157 PT8 were tested by MLVA; 39% were received in the first 4 months of 2011, when infections are generally low. One profile, or single locus variants of it, was present in 249 (52%) isolates but was not common previously. These cases represented a national increase in PT8, associated epidemiologically with soil-contaminated vegetables. Most of the 177 other MLVA profiles were unique to a single isolate. Profiles shared by >1 isolate included cases from two small community, food-borne outbreaks and 11 households. Several shared profiles were found among 23 isolates without known links. Apart from one group, isolates linked to travel abroad had very diverse profiles. CONCLUSIONS: Multilocus variable number tandem repeat analysis discriminated apparent sporadic isolates of the same PT and assisted in detection of cases in an emerging national outbreak. SIGNIFICANCE AND IMPACT OF THE STUDY: Multilocus variable number tandem repeat analysis is an epidemiologically valid complement to surveillance and applicable as a rapid, practical test for large numbers of isolates.

Ongoing outbreak of Shigella flexneri serotype 3a in men who have sex with men in England and Wales, data from 2009-2011.

Diagnoses of Shigella flexneri in the United Kingdom (UK) are usually travel-related. However, since 2009, there has been an overall increase in UK-acquired cases. The Health Protection Agency has been investigating a national outbreak of S. flexneri detected in 2011 and which is still ongoing. Cases occurred mostly in men who have sex with men and were of serotype 3a. The investigation aimed at obtaining epidemiological data to inform targeted outbreak management and control.

Shiga toxin-producing Escherichia coli O157 associated with human infections in Switzerland, 2000-2009.

Shiga toxin-producing Escherichia coli (STEC), an important foodborne pathogen, can cause mild to severe bloody diarrhoea (BD), sometimes followed by life-threatening complications such as haemolytic uraemic syndrome (HUS). A total of 44 O157 strains isolated from different patients from 2000 through 2009 in Switzerland were further characterized and linked to medical history data. Non-bloody diarrhoea was experienced by 15.9%, BD by 61.4% of the patients, and 29.5% developed HUS. All strains belonged to MLST type 11, were positive for stx2 variants (stx2 and/or stx2c), eae and ehxA, and only two strains showed antibiotic resistance. Of the 44 strains, nine phage types (PTs) were detected the most frequent being PT32 (43.2%) and PT8 (18.2%). By PFGE, 39 different patterns were found. This high genetic diversity within the strains leads to the conclusion that STEC O157 infections in Switzerland most often occur as sporadic cases.

Verocytotoxigenic Escherichia coli O157 in animals on public amenity premises in England and Wales, 1997 to 2007.

At the request of the public health authorities, 31 public amenity premises in England and Wales containing animals of various species were investigated for the presence of verocytotoxigenic Escherichia coli (VTEC) O157 between 1997 and 2007, because of putative associations with human cases. VTEC O157 was confirmed in one or more species on 19 (61.3 per cent) of the premises. There were significant associations between the presence of VTEC O157 and the number of species sampled, the size of the enterprise, the presence of young cattle and the presence of adult pigs. E coli O157 was isolated from 305 (17.8 per cent) of 1715 samples taken from all the premises, and verocytotoxin genes were detected by PCR in 184 (98.4 per cent) of 187 representative isolates. On positive premises, the highest mean proportion of positive samples (29.0 per cent) was in cattle, followed by sheep (24.4 per cent), donkeys (14.6 per cent), pigs (14.3 per cent), horses (12.3 per cent) and goats (9.9 per cent). A high proportion of positive samples was obtained from camelid species sampled on three of the premises. The main phage types (PT) were 2 and 21/28, which were those most commonly isolated from human cases during the same period. A single PT was detected on 14 of the 19 positive premises, with up to six different species having the same PT.

Intestinal carriage of verocytotoxigenic Escherichia coli O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica, in cattle, sheep and pigs at slaughter in Great Britain during 2003.

An abattoir survey was undertaken to determine the prevalence of foodborne zoonotic organisms colonizing cattle, sheep and pigs at slaughter in Great Britain. The study ran for 12 months from January 2003, involved 93 abattoirs and collected 7703 intestinal samples. The design was similar to two previous abattoir surveys undertaken in 1999-2000 allowing comparisons. Samples were examined for VTEC O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. The prevalence of VTEC O157 faecal carriage was 4.7% in cattle, 0.7% in sheep and 0.3% in pigs. A significant decrease in sheep was detected from the previous survey (1.7%). Salmonella carriage was 1.4% in cattle, a significant increase from the previous survey of 0.2%. In sheep, faecal carriage was 1.1% a significant increase from the previous survey (0.1%). In pigs, carriage was 23.4%, consistent with the previous study. Thermophilic Campylobacter spp. were isolated from 54.6% of cattle, 43.8% of sheep and 69.3% of pigs. Y. enterocolitica was isolated from 4.5% of cattle, 8.0% of sheep and 10.2% of pigs.

Association of putative pathogenicity genes with adherence characteristics and fimbrial genotypes in typical enteroaggregative Escherichia coli from patients with and without diarrhoea in the United Kingdom.

The aim of this study was to compare genotypic characteristics seen in typical EAggEC isolated during a study of intestinal infectious disease from cases and controls, and to identify which genes, or combinations of genes, were most associated with diarrhoeal disease. We also investigated the association of genotype with certain characteristics, such as presence of fimbrial genes and adherence to Hep-2 cells. The aafC gene, encoding the usher for AAFII, was the only gene significantly associated with patients with diarrhoea (P < 0.005), and the aggC gene, which encodes the usher for AAFI, was the only gene significantly associated with the healthy control group (P < 0.002). Putative virulence genes significantly associated with aggregative adherence included aafC, aggR, pet, pic and astA. The shf, pet and astA genes were all more likely to be associated with type II fimbriae than with type I. We conclude that in addition to presence and absence of certain genes, studies of EAggEC pathogenicity should investigate the combinations and associations of putative virulence factors.

Human Vero cytotoxigenic Escherichia coli (VTEC) O157 infection linked to birds.

Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) infections are a threat to public health. VTEC O157 has been isolated from gulls but evidence of transmission to humans from birds has not been reported. We recount an incident of VTEC O157 infection affecting two sibling children who had no direct contact with farm animals. An outbreak control team was convened to investigate the source of infection, its likely mode of transmission, and to advise on control measures. Human and veterinary samples were examined and the human isolates were found to be identical to an isolate from a sample of bird (rook) faeces. Cattle, rabbit and environmental samples were negative. This report provides evidence that birds may act as intermediaries for human infection with VTEC O157.

Serotypes, intimin subtypes, and antimicrobial resistance patterns of atypical enteropathogenic Escherichia coli isolated in England from 1993 to 1996.

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 general outbreaks of Shiga toxin-producing Escherichia coli O157 in England and Wales 1992-2002: where are the risks?

Between 1 January 1992 and 31 December 2002, Shiga toxin-producing Escherichia coli O157 (STEC O157) accounted for 44 of the 1645 foodborne general outbreaks of infectious intestinal disease reported to the Health Protection Agency Communicable Disease Surveillance Centre. These outbreaks, although rare, were characterized by severe infection, with 169 hospital admissions and five deaths reported. STEC O157 outbreaks were compared with other pathogens to identify factors associated with this pathogen. Single risk variable analysis and logistic regression were employed. Two distinct aetiologies were identified. Foodborne outbreaks of STEC O157 infection in England and Wales were independently associated with farms, which related to milk and milk products, and with red meats/meat products, which highlighted butchers' shops as a cause for concern. The introduction and adherence to effective control measures, based on the principles of hazard analysis, provide the best means of minimizing the risk of foodborne infection with this pathogen.

Shedding patterns of verocytotoxin-producing Escherichia coli strains in a cohort of calves and their dams on a Scottish beef farm.

Rectal 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-producing Escherichia 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 the eae gene. 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 the vtx1, eae, and ehxA genes, whereas vtx2 was associated with VTEC O2 and provisional serogroup E40874. The high prevalence of VTEC O26 and of VTEC strains harboring the eae gene in this calf cohort is notable because of the association of the O26 serogroup and the presence of the eae gene with human disease. No association between calf diarrhea and any of the VTEC serogroups was identified.

Increasing incidence of resistance to nalidixic acid in shigellas from humans in England and Wales: implications for therapy.

Among shigellas isolated from patients in England and Wales in 2002, 10% of subgroups A, B and C, and 13% of subgroup D (Shigella sonnei), were resistant to nalidixic acid. As a consequence, should antimicrobial therapy be indicated, the efficacy of nalidixic acid as the preferred treatment for children with bacillary dysentery has been jeopardised.

Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK.

OBJECTIVES: During 2003, the Health Protection Agency's Antibiotic Resistance Monitoring and Reference Laboratory began to receive isolates of Escherichia coli for confirmation of extended-spectrum beta-lactamase production with a phenotype implying a CTX-M-type beta-lactamase, i.e. MICs of cefotaxime > or = 8-fold higher than MICs of ceftazidime. Many were referred as being from community patients. We examined 291 CTX-M-producing isolates from the UK and investigated the genetic basis of their phenotype. METHODS: PCR was used to detect alleles encoding CTX-M enzymes and to assign these to their blaCTX-M phylogenetic groups. Selected alleles were sequenced. Producers were compared by analysis of banding patterns generated by pulsed-field gel electrophoresis of XbaI-digested genomic DNA. MICs were determined by an agar dilution method or by Etest. RESULTS: Of 291 CTX-M-producing E. coli isolates studied from 42 UK centres, 70 (24%) were reportedly from community patients, many of whom had only limited recent hospital contact. Community isolates were referred by 12 centres. Two hundred and seventy-nine (95.9%) producers contained genes encoding group 1 CTX-M enzymes and 12 contained blaCTX-M-9-like alleles. An epidemic CTX-M-15-producing strain was identified, with 110 community and inpatient isolates referred from six centres. Representatives of four other major strains also produced CTX-M-15, as did several sporadic isolates examined. Most producers were multi-resistant to fluoroquinolones, trimethoprim, tetracycline and aminoglycosides as well as to non-carbapenem beta-lactams. CONCLUSIONS: CTX-M-producing E. coli are a rapidly developing problem in the UK, with CTX-M-15 particularly common. The diversity of producers and geographical scatter of referring laboratories indicates wide dissemination of blaCTX-M genes. Because of the public health implications, including for the treatment of community-acquired urinary tract infections, the spread of these strains--and CTX-M-15 beta-lactamase in particular--merits close monitoring.

Comparison of the biotypes of Yersinia enterocolitica isolated from pigs, cattle and sheep at slaughter and from humans with yersiniosis in Great Britain during 1999-2000.

AIMS: To investigate the relationship between livestock carriage of Yersinia enterocolitica and human disease. The biotypes/serotypes of strains recovered from the faeces of pigs, cattle and sheep at slaughter during a national survey in Great Britain in 1999-2000, were compared with those of strains isolated from human cases of yersiniosis during the same period. METHODS AND RESULTS: The faecal carriage of Y. enterocolitica by cattle, sheep and pigs at slaughter was 6.3, 10.7 and 26.1%, respectively. Yersinia enterocolitica biotype (BT) 1a was the most frequently isolated biotype from livestock (58%) and was the predominant biotype (53%) isolated from human cases over the same period. The main recognized pathogenic Y. enterocolitica biotype isolated from livestock was BT3 (O:5,27) (35% of sheep, 22% of pigs and 4% of cattle) but this biotype was not detected in any of the human isolates investigated. The major pathogenic biotypes of strains isolated from humans were BT3 (O:9) (24%) and BT4 (O:3) (19%) whereas of the veterinary isolates investigated, only pigs (11%) carried BT3 (O:9) strains. CONCLUSIONS: Because of significant overlaps in phenotypes of the veterinary and human strains it is not possible to comment on the correlation between host and pathogenicity, especially of biotype 1a. SIGNIFICANCE AND IMPACT OF THE STUDY: The data suggest that further investigations using methods with greater discriminatory power are required. However the data also suggests that pigs may be the primary reservoir for human pathogenic Y. enterocolitica infection.

Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26, O103, O111, O145, and O157 in a cohort of beef calves and their dams.

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.

DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources.

AIMS: To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2. METHODS AND RESULTS: Eleven human and food strains isolated over a 15-year period were examined. All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant. Ten strains had VT2 genes which were all vtx2d. Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory. Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. CONCLUSIONS: As a result of apparent clonality, PFGE proved essential for differentiation. Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.

Prevalence of faecal excretion of verocytotoxigenic Escherichia coli O157 in cattle in England and Wales.

During the decade to 1999, the incidence of human infections with the zoonotic pathogen verocytotoxin-producing Escherichia coli O157 (VTEC O157) increased in England and Wales. This paper describes the results of a survey of 75 farms to determine the prevalence of faecal excretion of VTEC O157 by cattle, its primary reservoir host, in England and Wales. Faecal samples were collected from 4663 cattle between June and December 1999. The prevalence of excretion by individual cattle was 4.2 per cent (95 per cent confidence interval [CI] 2.0 to 6.4) and 10.3 per cent (95 per cent CI 5.8 to 14.8) among animals in infected herds. The within-herd prevalence on positive farms ranged from 1.1 to 51.4 per cent. At least one positive animal was identified on 29 (38.7 per cent; 95 per cent CI 28.1 to 50.4) of the farms, including dairy, suckler and fattening herds. The prevalence of excretion was least in the calves under two months of age, peaked in the calves aged between two and six months and declined thereafter. The phage types identified most widely were 4, 34 and 2, which were each found on six of the 29 positive farms.

Subtyping of virulence genes in verocytotoxin-producing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United Kingdom.

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.

Subtyping intimin genes from enteropathogenic Escherichia coli associated with outbreaks and sporadic cases in the United Kingdom and Eire.

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 alpha, beta, gamma, delta and zeta-intimin were detected in the EPEC strains associated with outbreaks and beta, gamma, epsilon, theta and zeta-intimin genes were identified in isolates from sporadic cases. The beta-intimin gene was the most frequently detected sub-type in both the outbreak and sporadic strains.