G. Frankel

Enterohaemorrhagic escherichia coli O157:H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine

BACKGROUND EnterohaemorrhagicEscherichia coli (EHEC) constitute a significant risk to human health worldwide, and infections, particularly with serogroup O157:H7, are associated with consumption of a variety of food and water vehicles, particularly food of bovine origin. EHEC cause acute gastroenteritis, bloody diarrhoea, and haemorrhagic colitis; up to 10% of cases develop severe complications, including the haemolytic uraemic syndrome, with a 5% case fatality. A virulence characteristic of enteropathogenic E coli, the attaching/effacing lesion, is considered to be important in EHEC. However, although EHEC produce this lesion on cultured human cells, this has not been demonstrated on human intestinal mucosal surfaces. In addition, the initial site(s) of colonisation of EHEC in humans is not known. AIMS To assess the association of EHEC O157:H7 with paediatric and bovine intestine using in vitro organ culture and determine if attaching/effacing lesions occur. METHODS Ultrastructural analysis of in vitro intestinal organ cultures of human small and large intestine was used to investigate adhesion of O157:H7 EHEC to intestinal surfaces. Bovine intestinal organ culture was used to examine the pathology produced by the same EHEC strain in cattle. RESULTS The study showed that EHEC O157:H7 adhered to human intestinal mucosa. Binding and attaching/effacing lesion formation of O157:H7 in humans was restricted to follicle associated epithelium of Peyers patches. The same strain caused attaching/effacing lesions on bovine mucosa. CONCLUSIONS O157:H7 targets follicle associated epithelium in humans where it causes attaching/effacing lesions. The same human isolate can cause attaching/effacing lesions in cattle, indicating that similar pathogenic mechanisms operate across human and bovine species

Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques.

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.

Characterization of Two Non-Locus of Enterocyte Effacement-Encoded Type III-Translocated Effectors, NleC and NleD, in Attaching and Effacing Pathogens

ABSTRACT Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.

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.

Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhoea in Germany.

Aims:  To investigate the prevalence of traditional and emerging types of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) strains in stool samples from children with diarrhoea and to characterize their virulence genes involved in the attaching and effacing (A/E) phenotype.

Tissue Tropism of Enteropathogenic Escherichia coli Strains Belonging to the O55 Serogroup

ABSTRACT Four enteropathogenic Escherichia coli (EPEC) strains belonging to the O55 serogroup (G21 and G30 [both O55:H6], G35 [O55:H−], and G58 [O55:H7]) were tested for their tissue tropism by using human intestinal in vitro organ culture. Strains showed restricted adhesion with attaching-and-effacing activity to follicle-associated epithelium of Peyers patches, with no apparent adhesion to duodenum or colon. G35 and G58 express intimin γ and show a similar tropism to intimin γ-expressing enterohemorrhagic E. coli (EHEC) O157:H7. However, strains G21 and G30 were unusual because they expressed intimin α and had a restricted tissue tropism of intimin γ phenotype. The amino acid sequence of the carboxy-terminal 280 amino acids of intimin from G21 was determined. Comparison with the prototype intimin α from strain E2348/69 (O127:H6) showed a single amino acid difference (corresponding to Val907 and Ala907 in the whole intimins). This mutation was reproduced by site-directed mutagenesis in an intimin α plasmid template, pCVD438, with the hypothesis that it may induce a change in tropism. However, when the mutated plasmid was placed in both EPEC and EHEC backgrounds, duodenal adhesion in a manner similar to strain E2348/69 was evident upon in vitro organ culture. Thus, additional factor(s) unrelated to intimin exist in the O55:H6 genome that influence human intestinal tissue tropism.

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.

Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants

Background:Salmonella enterica serovar typhimurium (Styphimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. Aims: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). Methods: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. Results:S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.Styphimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading Styphimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. Conclusions: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis.

An eight-month study of a population of verocytotoxigenic Escherichia coli (VTEC) in a Scottish cattle herd

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.

Enhanced Susceptibility to Citrobacter rodentium Infection in MicroRNA-155-Deficient Mice

ABSTRACT MicroRNAs (miRNAs) are small noncoding molecules that control gene expression posttranscriptionally, with microRNA-155 (miR-155) one of the first to be implicated in immune regulation. Here, we show that miR-155-deficient mice are less able to eradicate a mucosal Citrobacter rodentium infection than wild-type C57BL/6 mice. miR-155-deficient mice exhibited prolonged colonization associated with a higher C. rodentium burden in gastrointestinal tissue and spread into systemic tissues. Germinal center formation and humoral immune responses against C. rodentium were severely impaired in infected miR-155-deficient mice. A similarly susceptible phenotype was observed in μMT mice reconstituted with miR-155-deficient B cells, indicating that miR-155 is required intrinsically for mediating protection against this predominantly luminal bacterial pathogen.