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.

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

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

A simple method for the preparation of large quantities of pure plasmid DNA.

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Strains of Escherichia coli O157:H8 from human diarrhoea belong to attaching and effacing class of E coli.

AIMS: To determine whether 17 Escherichia coli O157:H8 strains isolated from patients with diarrhoea in the United Kingdom were putative pathogens. METHODS: The strains had been isolated by the use of O157 antiserum, available for the detection of Vero cytotoxin (VT) producing strains of E coli O157 that are usually of flagellar (H) type 7, but may also be non-motile. The strains were examined for VT production, for their ability to adhere to HEp-2 cells, and for hybridisation with several DNA probes that recognise pathogenic properties of E coli. Their ability to ferment sorbitol and to produce beta-glucuronidase was also investigated, as these tests are used to discriminate VT positive O157 strains. RESULTS: The O157:H8 strains did not produce VT. All gave localised attachment to HEp-2 cells, associated with a positive fluorescence-actin staining test, and all hybridised with the E coli attaching and effacing (eae) probe. In addition to the difference in VT production, O157:H8 strains could be distinguished from VT positive O157 strains by their beta-glucuronidase activity, their failure to produce enterohaemolysin, and their lack of hybridisation with the CVD419 probe derived from a plasmid in an O157:H7 strain. CONCLUSIONS: The 0157:H8 strains had in vitro properties characteristic of the class of E coli that causes attaching and effacing lesions in epithelial intestinal cells. They may therefore be considered a putative cause of diarrhoea but their prevalence remains to be established. Several O157:H8 strains failed to ferment sorbitol in agar plates and therefore could be misidentified as VT positive O157 strains. Confirmatory tests for VT production are needed when O157 strains are isolated from faeces.

Cloning of regulator genes controlling fimbrial production by enterotoxigenic Escherichia coli.

Sequences regulating production of fimbriae were cloned from two enterotoxigenic Escherichia coli strains. One cloned region, from E. coli 0.25.H42, controlled expression of coli surface-associated (CS) antigen 4, whereas the function of the other, from E. coli 0167.H5, was unclear. Both regulators were related to the cfaD gene that controls expression of colonization factor antigen I (CFA/I) although low stringency conditions were required to show significant hybridization between cfaD and the regulatory fragment from E. coli 0167. The cloned regulatory genes promoted expression of CFA/I, CS1, CS2 and CS4 antigens but the levels of production in the presence of the 0167 regulator were lower than those promoted by the CS4 regulator or cfaD.

Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 0.25.H42.

Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I. Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6. By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen. The structural genes for the CS4 antigen were on a separate plasmid. The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.

The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5.

A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.

Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139:H28.

An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon. The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined. Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found. The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon. For all the CS1 producing strains investigated the structural genes are located on plasmids. Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively. The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267. PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD.

The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits.

The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes. The DNA sequences from both clones were nearly identical and contained four open reading frames. Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors. The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit. Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.

Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe.

Strains of Escherichia coli from sporadic cases of diarrhoea and belonging to serotypes O44:H18, O55:H7, O111ab:H21, O111ab:H25 or O126:H27 were examined for virulence properties. With the exception of O111ab:H25 these are considered to be classical enteropathogenic E. coli (EPEC) serotypes. The strains had been isolated in Britain from the faeces of children less than 3 years old. Of the serotypes examined, 7 of 13 O44:H18 strains, all of 10 O111ab:H21 strains and 13 of 21 O126:H27 strains belonged to the enteroaggregative class of E. coli (EAggEC) that attached to HEp-2 cells in the characteristic aggregative pattern and hybridised with the EAggEC probe. They also caused mannose-resistant haemagglutination of rat erythrocytes, a property which may be a useful marker for their identification. Strains of O44:H18 with similar properties were also isolated from three small outbreaks in Britain, one of which involved elderly patients. EAggEC have not been considered previously as aetiological agents of diarrhoea in developed countries and have rarely been reported as belonging to EPEC serotypes. All 15 O55:H7 strains and seven of eight O111ab:H25 strains were also considered to be potentially diarrhoeagenic as they gave localised attachment (LA) to HEp-2 cells that resulted in a positive fluorescence actin-staining test. This test is considered to correlate with the attaching-and-effacing virulence mechanisms of EPEC in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Vero cytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes.

Faecal specimens from 66 children with haemolytic uraemic syndrome in the United Kingdom were examined for strains of Escherichia coli producing Vero cytotoxin (VT). Initially, conventional bacteriological methods were used to identify colonies of E. coli which were then tested for VT production. Subsequently, specific DNA probes for VT1 and VT2 were used in hybridisation tests to detect VT-producing E. coli (VTEC). VTEC strains were isolated from 19 cases and in 15 they belonged to serogroup O157. Fourteen of these O157 strains possessed the flagellar antigen H7 and one was non-motile. The VTEC strains from the remaining four cases belonged to serotypes O26:H11, O104:H2, O153:H25, and O163:H19 together with a rough VT+ strain with flagellar antigen H51. The O157 strains hybridised with either the VT2 probe or both VT1 and VT2 probes. The other VTEC strains hybridised with either the VT1 or VT2 probe. Confirmation of the production of VT1 and VT2 in vivo was obtained by the neutralisation of faecal VT with specific antisera raised against these two cytotoxins.

Verocytotoxin-producing Escherichia coli (VTEC) O157 and other VTEC from human infections in England and Wales: 1995-1998.

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.

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.

Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom.

Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E. coli (EPEC) O serogroups were examined for virulence properties. The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E. coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E. coli and diffusely adherent E. coli. The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142. Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes. Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive. However, <10% of these eae probe-positive strains hybridised with the EAF probe. Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative. VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128. Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties.

Vero cytotoxin-producing Escherichia coli in a study of infectious intestinal disease in England.

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.

Applications of DNA probes for Vero cytotoxin-producing Escherichia coli.

Vero cytotoxin-producing Escherichia coli (VTEC) are now recognized as important aetiological agents in human disease. The symptoms of VTEC infection range from mild non-bloody diarrhoea to severe conditions such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Two types of Vero cytotoxin (VT), VT1 and VT2, have been identified. The genes controlling production of VT1 and VT2 are phageencoded in several E. coli strains and DNA probes have been developed from cloned genes derived from these VT phages. Recently, synthetic oligonucleotide probes for VTEC have also been prepared and evaluated. The VT probes have been labelled radioactively and also non-radioactively with digoxigenin and biotin. Present applications of VT probes include detection of VTEC in faecal samples from patients with diarrhoea, HC or HUS and also the examination of different foods for the presence of VTEC. The proportion of VTEC in the faecal flora or foods may be low, often less than 1%. The use of DNA probes allows several hundred colonies from a sample to be examined and by this technique VT genes were detected in 30-40% of faecal specimens from patients with HC or HUS. Use of methods such as the polymerase chain reaction for amplification of the target genes combined with DNA probes should result in an increased sensitivity for the detection of VTEC.

The emergence and spread of antibiotic resistance in food-borne bacteria.

Since the early 1990s there has been a dramatic increase in resistance to antimicrobial drugs in Salmonella enterica and Campylobacter spp., and to a lesser extent in Vero cytotoxin-producing Escherichia coli O157 from cases of human infection in developed countries. For S. Typhimurium a particularly important aspect of this increase has been the widespread dissemination of a multiply drug-resistant (MR) strain of definitive phage type (DT) 104 in food animals since the early 1990s. The use of antimicrobials for prophylaxis in food producing animals has been an important factor in the emergence of strains with resistance to certain antimicrobials. It is hoped that recently introduced Codes of Practice for the prophylactic use of antimicrobials in food animals will result in a decline in the occurrence of drug resistant strains in the food chain.

Use of strain typing to provide evidence for specific interventions in the transmission of VTEC O157 infections.

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.

Adhesion to cells in culture and plasmid profiles of enteropathogenic Escherichia coli isolated from outbreaks and sporadic cases of infant diarrhoea.

Enteropathogenic strains of Escherichia coli (EPEC) that caused 10 outbreaks of infant diarrhoea in the U.K. between 1968 and 1986 were studied. All gave localised adherence (LA) to HEp-2 cells, HeLa cells and Intestine 407 cells in culture. All hybridised with the EPEC adherence factor (EAF) probe. The hybridising sequences were carried on plasmids ranging in size from 26 to 76 MDa. EPEC from sporadic cases of infant diarrhoea occurring between 1979 and 1986 that belonged to the same serotypes as the outbreak strains were also studied. All strains of serotypes O111ab.H2, O114.H2, O119.H6, O127.H6 and O142.H6 gave LA and were EAF-positive. In other serotypes, non-adhering strains or strains giving diffuse adherence were found also. In addition, strains of serotype O128.H2 which gave LA but did not hybridise with the EAF probe were identified. The strains isolated from sporadic cases of diarrhoea in the U.K. were similar, with respect to adhesion and hybridisation, to those isolated from sporadic cases of diarrhoea in developing countries.

Phage typing and DNA-based comparison of strains of enterohemorrhagic Escherichia coli O157 from apparently sporadic infections in Osaka City, Japan, 1996.

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.