When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Veterinary Research, a journal on Animal Infection

EnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coli(EHEC) produce the characteristic “attaching and effacing” (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded byeae, is responsible for the tight association of both pathogens with the host cell. Severaleaehave been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate theeaeheterogeneity of the variable 3′ region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains wereeaepositive when primers amplifying the conserved 5′ end were used. We then cloned and sequenced theeaepresent in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3′ region of this new intimin type. This intimin, referred to as “ɛ,” was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin ɛ is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin β, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins α and γ.

We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx _2f . This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx _2f gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.

In this study, we determined the sequences of four intimin variant genes detected in attaching and effacingEscherichia coliisolates of human origin. Three of them were novel and were designatedeae-η (eta),eae-ι (iota), andeae-κ (kappa). The fourth was identical to the recently describedeae-ζ (zeta), isolated from a bovineE. coliO84:NM isolate. We compared these sequences with those of published intimin-α, intimin-β, intimin-γ1, intimin-γ2, intimin-ε, and intimin-θ alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family inE. coli.The genetic diversity was more prominent in the 3′ region (starting at bp 2112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: α and ζ; β and κ; γ1 and γ2/θ; and ε and η. Calculation of homoplasy ratios of sequences of the 5′ region ofeae(positions 1 to 2111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history ofeae.In conclusion, we recommend aneaenomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing ofE. coli eae.

The Escherichia coli strain causing a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in Germany in May and June 2011 possesses an unusual combination of pathogenic features typical of enteroaggregative E. coli together with the capacity to produce Shiga toxin. Through rapid national and international exchange of information and strains the known occurrence in humans was quickly assessed. We describe simple diagnostic screening tools to detect the outbreak strain in clinical specimens and a novel real-time PCR for its detection in foods.