Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli.

The European Union Reference Laboratory (EU-RL) has produced guidelines for a real-time PCR for the detection of verocytotoxigenic Escherichia coli (VTEC). In this study, we validated the EU-RL assay on 545 strains of VTEC and evaluated the utility of the assay for the detection of VTEC from stool specimens. The validation study on cultures showed that the EU-RL VTEC PCR was 99.3% sensitive for the detection of vtx genes; only strains harbouring vtx2f genes were not detected. The assay was 100% sensitive and 100% specific for the detection of both the eae and O157 rfbE genes. In a prospective study involving 500 stool samples, the EU-RL VTEC PCR detected vtx genes in 12.4% of specimens, compared to 3.8% specimens found to be culture-positive for E. coli O157 using the Health Protection Agency national standard culture method. This study showed that the EU-RL VTEC assay was reliable and robust, and an effective rapid screening method for the detection of VTEC from stool specimens.

UK epidemic Escherichia coli strains A-E, with CTX-M-15 beta-lactamase, all belong to the international O25:H4-ST131 clone.

OBJECTIVES: Uropathogenic and invasive Escherichia coli O25:H4-ST131 isolates producing CTX-M-15 extended-spectrum beta-lactamase (ESBL) enzymes have recently been shown to be disseminated across the globe. In the UK, many CTX-M-15 ESBL-producing E. coli strains have been previously defined as belonging to the epidemic strains A-E, as determined by PFGE. The present study was carried out to define the relationship between these two groups of pathogenic E. coli. METHODS: Multilocus sequence typing and PFGE were used for molecular characterization of a collection of 61 ESBL-producing E. coli isolates from across the UK. RESULTS: Strains A to E all belonged to the ST131 clone, further underscoring the epidemiological importance of this lineage. CONCLUSIONS: The future spread of the ST131 clone, and its UK variants, should be monitored closely and the pathogenic mechanisms explaining their success should be investigated.

Detection of enteroaggregative Escherichia coli in faecal samples from patients in the community with diarrhoea.

The aim of this study was to assess the usefulness of a multiplex PCR assay targeting the aat, aaiA and astA genes for the detection of typical and atypical enteroaggregative Escherichia coli (EAEC) in bacterial cultures from faecal samples from patients with community-acquired diarrhoea. The isolates harbouring these genes were also tested using the HEp-2 cell-adhesion assay to clarify their EAEC status. aat, aai or astA was found in E. coli faecal isolates from 39 (7.8 %) of 500 patients, and 20 of these strains adhered to HEp-2 cells in a pattern characteristic of EAEC. Eight isolates carrying the aai or astA gene but not the aat gene were shown to be HEp-2 cell test positive, although 12 strains with this genotype were HEp-2 cell test negative. Using the HEp-2 adhesion assay as the gold standard, the addition of primers detecting aaiA and astA to the aat PCR increased the number of EAEC isolates detected, but identified strains of E. coli that were not EAEC. The variety of genotypes exhibiting aggregative adherence highlights the problems associated with developing a molecular diagnostic test for EAEC. This PCR assay detects a variety of strains exhibiting characteristics of the EAEC group, making it a useful tool for identifying both typical and atypical EAEC.

Genotyping of enteroaggregative Escherichia coli and identification of target genes for the detection of both typical and atypical strains.

Enteroaggregative Escherichia coli (EAggEC) is an important cause of diarrhea worldwide, and there is a need for better detection methods in diagnostic laboratories. The aims of this study were i) to characterize strains of EAggEC by assigning each isolate a genotypic profile and (ii) to determine target genes for the detection of both typical and atypical EAggEC. The heterogeneity of the EAggEC group makes selection of a single target gene difficult. The plasmid-encoded genes, aat, aggR, and aap, are all appropriate targets for the detection of typical EAggEC. Of the chromosomally encoded genes, aaiA would be the most suitable target gene to identify typical and atypical EAggEC. The astA gene, encoding the enteroaggregative heat stable toxin, although not specific for EAggEC, may be used effectively in combination with other specific EAggEC genes. A polymerase chain reaction test based on the detection of characteristic EAggEC virulence genes, such as aat, astA, and aaiA, would improve EAggEC diagnosis.

Use of a microarray to assess the distribution of plasmid and chromosomal virulence genes in strains of enteroaggregative Escherichia coli.

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.

Childhood hemolytic uremic syndrome, United Kingdom and Ireland.

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.

Analysis of saliva for antibodies to the LPS of Escherichia coli O157 in patients with serum antibodies to E. coli O157 LPS.

The salivary antibody response to the Escherichia coli O157 LPS antigen was assessed in 44 patients with serum antibodies binding to the LPS of E. coli O157. Saliva from 477 controls was also examined to assess the specificity of the immunoassay used. Twenty of the 44 patients had salivary antibodies to E. coli O157 LPS, giving the salivary antibody test a sensitivity of 0.45 and a predictive positive value for seropositivity of 1.00. The presence of these antibodies appeared not to relate to the time interval between serum sampling and saliva sampling. None of the 477 volunteers had salivary antibodies binding to the LPS of E. coli O157 alone; however, 15 had antibodies which bound non-specifically to both O157 LPS and BSA.

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli.

Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.

Characteristics and association with disease of two major subclones of Shiga toxin (Verocytotoxin)-producing strains of Escherichia coli (STEC) O157 that are present among isolates from patients in Germany.

Shiga toxin (Verocytotoxin) producing E. coli (STEC) O157 were isolated from 168 patients living in different parts of Germany. Most isolates were from sporadic cases and seven small outbreaks with STEC O157 were identified. The 168 strains were examined for phenotypic and genotypical traits in order to identify major types of STEC O157 occurring in Germany. Phage typing (PT) revealed PT8 (n = 54) and PT2 (n = 48) strains as most frequent (60.7%) among the isolates. Carriage of the stx(2) gene by STEC O157 was closely associated with hemolytic uremic syndrome (100%) and with bloody diarrhea (61.7%). The stx(2) gene was frequent in PT88, PT47 (both 100%), PT2 (91.5%) and PT4 (87.5%) strains and more rarely (33.3%) found in strains belonging to the other PTs. PT8 and PT2 strains formed two groups which differed from each other in their motility, stx-genotypes and the severity of the illness they caused. Pulsed-field gel electrophoresis of PT2 and PT8 strains and hybridization of XbaI digested DNA with stx(1) and stx(2) specific gene probes revealed similarities among epidemiologically unrelated strains belonging to the same PT. The results indicate that STEC O157 PT2 and PT8 strains form two distinct subclones which are dominating in Germany and other European countries.