Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle

Multilocus-genotyping methods have shown that Escherichia coli O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demonstrate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of E. coli O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance between over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning products derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of E. coli O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restriction fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explanation for the degree of diversity that is observed among E. coli O157:H7 by other molecular-fingerprinting methods.

Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins

Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of E. coli O157:H7. We were interested in knowing why cattle are tolerant to infection with E. coli O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb3), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo. TLC was used to detect Gb3 in tissues from a newborn calf. Gb3 was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb3 and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable E. coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E. coli O157:H7.

The prevalence and concentration of Escherichia coli O157 in faeces of cattle from different production systems at slaughter

Aims: To determine the prevalence and concentration of Escherichia coli O157 shed in faeces at slaughter, by beef cattle from different production systems. Methods and Results: Faecal samples were collected from grass-fed (pasture) and lot-fed (feedlot) cattle at slaughter and tested for the presence of E. coli O157 using automated immunomagnetic separation (AIMS). Escherichia coli O157 was enumerated in positive samples using the most probable number (MPN) technique and AIMS and total E. coli were enumerated using Petrifilm. A total of 310 faecal samples were tested (155 from each group). The geometric mean count of total E. coli was 5 x 10(5) and 2.5 x 10(5) CFU g(-1) for lot- and grass-fed cattle, respectively. Escherichia coli O157 was isolated from 13% of faeces with no significant difference between grass-fed (10%) and lot-fed cattle (15%). The numbers of E. coli O157 in cattle faeces varied from undetectable (

An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination

Several pathogenic strains of Escherichia coli exploit type III secretion to inject effector proteins into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of > 60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into > 20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in > 20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage metagenome, acting as a crucible for the evolution of pathogenicity.

Laboratory-based simulation of freezing profiles of beef trim for Escherichia coli O157 survival determinations

This study was undertaken to develop a simple laboratory-based method for simulating the freezing profiles of beef trim so that their effect on E. coli 0157 survival could be better assessed. A commercially available apparatus of the type used for freezing embryos, together with an associated temperature logger and software, was used for this purpose with a -80 degrees C freezer as a heat sink. Four typical beef trim freezing profiles, of different starting temperatures or lengths, were selected and modelled as straight lines for ease of manipulation. A further theoretical profile with an extended freezing plateau was also developed. The laboratory-based setup worked well and the modelled freezing profiles fitted closely to the original data. No change in numbers of any of the strains was apparent for the three simulated profiles of different lengths starting at 25 degrees C. Slight but significant (P < 0.05) decreases in numbers (similar to 0.2 log cfu g(-1)) of all strains were apparent for a profile starting at 12 degrees C. A theoretical version of this profile with a freezing plateau phase extended from 11 h to 17 h resulted in significant (P < 0.05) decreases in numbers (similar to 1.2 log cfu g(-1)) of all strains. Results indicated possible avenues for future research in controlling this pathogen. The method developed in this study proved a useful and cost-effective way for simulating freezing profiles of beef trim. (c) 2005 Elsevier B.V. All rights reserved.

Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential of Salmonella enterica and Escherichia coli O157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for both S. enterica and E. coli O157. Cross-resistance in adaptively resistant S. enterica varied with the serotype; Salmonella enterica serovar Enteritidis expressed cross-resistance to chloramphenicol, whereas Salmonella enterica serovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistant Salmonella enterica serovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistant Salmonella serovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms. E. coli O157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.

Caractérisation du risque associé à la consommation de saucissons secs contaminés par Escherichia coli O157:H7

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Cortactin recruitment by enterohemorrhagic Escherichia coli O157:H7 during infection in vitro and ex vivo

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspF(U). Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

Caractérisation du risque associé à la consommation de saucissons secs contaminés par Escherichia coli O157:H7

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Identification des gènes de Escherichia coli entérohémorragique exprimés pendant l'infection de macrophages humains

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H- with a novel combination of virulence factors

Attaching and effacing (AE) lesions were observed in the caecum, proximal colon and rectum of one of four lambs experimentally inoculated at 6 weeks. of age with Escherichia coli O157:H7. However, the attached bacteria did not immunostain with O157-specific antiserum. Subsequent bacteriological analysis of samples from this animal yielded two E. coli O115:H- strains, one from the colon (CO) and one from the rectum (RC), and those bacteria forming the AE lesions were shown to be of the O115 serogroup by immunostaining. The O115:H(-)isolates formed microcolonies and attaching and effacing lesions, as demonstrated by the fluorescence actin staining test, on HEp-2 tissue culture cells. Both isolates were confirmed by PCR to encode the epsilon (epsilon) subtype of intimin. Supernates of both O115:H- isolates induced cytopathic effects on Vero cell monolayers, and PCR analysis verified that both isolates encoded EAST1, CNF1 and CNF2 toxins but not Shiga-like toxins. Both isolates harboured similar sized plasmids but-PCR analysis indicated that only one of the O115:H- isolates (CO) possessed the plasmid-associated virulence determinants ehxA and etpD. Neither strain possessed the espP, katP or bfpA plasmid-associated virulence determinants. These E. coli O115:H- strains exhibited a novel combination of virulence determinants and are the first isolates found to possess both CNF1 and CNF2.

Naturally acquired attaching and effacing Escherichia coli in sheep

In a series of experiments involving the inoculation of sheep with Escherichia coli O157:H7, and subsequent detailed histopathological examination of the intestinal mucosa, attaching-effacing (AE) lesions formed by elements of the natural flora were observed in 18% of animals. These incidental AE lesions typically were small and sparse, and were not associated with clinical disease. It was possible to identify further some of the lesional bacteria, revealing that E. coli O115 had formed lesions in one of the seven affected animals, and similarly E. coli O26 had formed some of the lesions in another. As AE strains, source flocks, housing and feed sources were diverse, a common source of lesion-forming bacteria appears to be unlikely. It is postulated that subclinical AE lesions are a mechanism of persistence of AE bacteria in sheep.