Factors in the emergence of serious human infections associated with highly pathogenic strains of shiga toxin-producing Escherichia coli.

The appearance of highly pathogenic strains of Shiga toxin (Stx)-producingEscherichia. coli (STEC) has owed largely to the acquisition of Stx-encoding prophages by strains of E. coli that have pre-existing potential as enteric pathogens, such as atypical enteropathogenic E. coli (aEPEC) and enteroaggregative E. coli (EAEC). However, while high pathogenic potential is necessary, it is not sufficient for such strains to have a serious public health impact (i.e., large outbreaks, many cases of HUS, or both). To do so requires susceptible hosts and additional elements related to transmission, such as, socio-economic, societal, and lifestyle, factors. Two examples are discussed to illustrate this. The factors involved in the emergence of serious disease associated with E. coli O157:H7 in the 1980s probably included a massive increase in population exposure to this pathogen, likely as a result of the introduction of factory farming of cattle in the 1960s, and the development and wide patronage of fast food hamburger restaurants, and, potentially, waning immunity to intimin as a result of the reduction of incidence of enteropathogenic E. coli (EPEC) infection. In the devastating outbreak of Stx2-positiveEAEC O104:H4 in 2011, the wide distribution of the proposed vehicle of transmission, imported fenugreek seeds, was decisive in the exposure of a large population in Central Europe to this pathogen. Contributing factors likely included a preference for eating raw sprouts as a healthy food choice by the affected cases, many of whom were women. Low population levels of immunity to Stx2 probably contributed to the severe clinical outcome. A better understanding of the factors responsible for the emergence of potentially dangerous STEC pathogens as well as of extensive and serious disease associated with them can enhance public health strategies to respond to them.

Emerging Public Health Challenges of Shiga Toxin-Producing Escherichia coli Related to Changes in the Pathogen, the Population, and the Environment.

Emerging public health challenges of Shiga toxin (stx)-producing Escherichia coli (STEC) include the occurrence of more frequent or severe disease and risk factors shifts associated with changes, often interconnected, in the pathogen, the population, and the environment. In 3 outbreaks with heightened severity attributed to enhanced pathogen virulence, including the acquisition of an stx2 phage in 1 outbreak, population and environmental factors likely contributed significantly to disease outcomes. Evolving population risk factors that are associated with more severe disease include consumption of fresh produce, contact with STEC-contaminated environments, demographics, socioeconomic status, and immunity. Risks of increasing STEC environmental pollution are related to continued intensification of agriculture and super-shedder cattle. Mitigation strategies include surveillance and research on emerging STEC, development of effective communications and public education strategies, and improved policies and interventions to mitigate risks, including those related to the contamination of produce and the environment, using a "One Health" approach.

Common variants of the vitamin D binding protein gene and adverse health outcomes.

The vitamin D binding protein (DBP) is the major plasma carrier for vitamin D and its metabolites, but it is also an actin scavenger, and is the precursor to the immunomodulatory protein, Gc-MAF. Two missense variants of the DBP gene - rs7041 encoding Asp432Glu and rs4588 encoding Thr436Lys - change the amino acid sequence and alter the protein function. They are common enough to generate population-wide constitutive differences in vitamin D status, based on assay of the serum metabolite, 25-hydroxyvitamin D (25OHD). Whether these variants also influence the role of vitamin D in an immunologic milieu is not known. However, the issue is relevant, given the immunomodulatory effects of DBP and the role of protracted innate immune-related inflammation in response to tissue injury or repeated infection. Indeed, DBP and vitamin D may jointly or independently contribute to a variety of adverse health outcomes unrelated to classical notions of their function in bone and mineral metabolism. This review summarizes the reports to date of associations between DBP variants, and various chronic and infectious diseases. The available information leads us to conclude that DBP variants are a significant and common genetic factor in some common disorders, and therefore, are worthy of closer attention. In view of the heightened interest in vitamin D as a public health target, well-designed studies that look simultaneously at vitamin D and its carrier in relation to genotypes and adverse health outcome should be encouraged.

Plasma 25-hydroxyvitamin D, hormonal contraceptive use, and cardiometabolic disease risk in an ethnically diverse population of young adults.

OBJECTIVES: The relationship between vitamin D and cardiometabolic disease risk across ethnic groups is unclear, and it is not known whether the use of hormonal contraceptives (HCs), which affect vitamin D metabolism and are also associated with cardiometabolic disease risk, modifies this relationship. Our objectives were to determine the prevalence of vitamin D deficiency (plasma 25-hydroxyvitamin D [25(OH)D] < 30 nmol/L) to assess seasonal variation in concentrations of 25(OH)D, and to examine whether 25(OH)D is associated with cardiometabolic biomarkers across ethnic groups and across men, female HC nonusers, and female HC users in an ethnically diverse population of young adults living in Canada. METHODS: The study population consisted of Caucasian, East Asian, and South Asian individuals (n = 1384, 69% female) aged 20-29 years. Participants provided overnight fasting blood samples, from which plasma 25(OH)D and cardiometabolic biomarkers were measured. Vitamin D status distributions were compared using χ(2) tests, and analysis of covariance (ANCOVA) was used to examine seasonal variations in 25(OH)D, as well as the association between 25(OH)D and cardiometabolic biomarkers, across groups. RESULTS: Plasma 25(OH)D concentrations fluctuated seasonally among Caucasians and East Asians and across men, female HC nonusers, and female HC users, but they remained low year-round in South Asians, half of whom were vitamin D deficient. Vitamin D deficiency was associated with higher insulin, homeostasis model assessment-estimated insulin resistance (HOMA-IR), and homeostasis model assessment (HOMA)-beta among Caucasians and East Asians and among men and female HC nonusers and with higher triglycerides among men only. No biomarkers were associated with 25(OH)D among South Asians and female HC users, although nonsignificant trends were observed for higher markers of glycemic dysregulation in those who were vitamin D deficient in both groups. CONCLUSIONS: Vitamin D deficiency varies between ethnic groups and is particularly high among South Asians, and it is associated with biomarkers of glycemic dysregulation; however, HC use among women may attenuate this association. Given the widespread use of HCs by women throughout the world, a better understanding of the extent to which these medications may modify the relationship between vitamin D and processes related to disease is warranted.

Plasma vitamin D and biomarkers of cardiometabolic disease risk in adult Canadians, 2007-2009.

INTRODUCTION: Vitamin D may modulate cardiometabolic disease risk, although the relationship has not been investigated in the general Canadian population. Understanding this relationship may inform public health strategies to curb the incidence of cardiometabolic disease in Canada and elsewhere. The objectives of this study were to examine the association between vitamin D and traditional and novel biomarkers of cardiometabolic disease and to describe the extent of the month-to-month fluctuations of vitamin D in the Canadian population. METHODS: We examined the association between plasma 25-hydroxyvitamin D and a range of cardiometabolic risk biomarkers in participants (n = 1,928; age range, 16-79 years) from the Canadian Health Measures Survey. We conducted linear regressions analyses (adjusted for sex, waist circumference, physical activity, hormone use, and season) to assess the relationship between 25-hydroxyvitamin D and biomarkers of dysglycemia, dyslipidemia, and inflammation in the study population. We repeated analyses stratified by sex, and we evaluated monthly fluctuations in 25-hydroxyvitamin D in men and women. RESULTS: We observed wide month-to-month variations in 25-hydroxyvitamin D; fluctuations were more pronounced in men. Plasma 25-hydroxyvitamin D was inversely associated with insulin, insulin resistance, triglycerides, total cholesterol, low-density lipoprotein cholesterol, and the ratio of total to high-density lipoprotein cholesterol but not with fasting glucose, apolipoprotein A1, apolipoprotein B, C-reactive protein, fibrinogen, or homocysteine. This pattern varied between men and women. CONCLUSION: Vitamin D may modulate various metabolic processes and may influence cardiometabolic disease risk in Canadians. These findings may have public health implications when recommending vitamin D for the prevention of cardiometabolic disease and related conditions.

Novel repressor of Escherichia coli O157:H7 motility encoded in the putative fimbrial cluster OI-1.

Escherichia coli O157:H7 is a gastrointestinal pathogen that has become a serious public health concern, as it is associated with outbreaks and severe diseases such as hemolytic-uremic syndrome. The molecular basis of its greater virulence than that of other serotypes is not completely known. OI-1 is a putative fimbria-encoding genomic island that is found almost exclusively in O157:H7 Shiga toxin-producing E. coli strains and may be associated with the enhanced pathogenesis of these strains. In this study, we identified and characterized a novel repressor of flagellar synthesis encoded by OI-1. We showed that deletion of Z0021 increased the motility of E. coli O157:H7, which correlated with an increase in flagellin production and enhanced assembly of flagella on the cell surface. In contrast, overexpression of Z0021 inhibited motility. We demonstrated that Z0021 exerted its regulatory effects downstream of the transcription and translation of flhDC but prior to the activation of class II/III promoters. Furthermore, the master regulator of flagellar synthesis, FlhD(4)C(2), was shown to be a high-copy suppressor of the nonmotile phenotype associated with elevated levels of Z0021--a finding consistent with Z0021-FlhD(4)C(2) being a potential regulatory complex. This work provides insight into the mechanism by which Z0021, which we have named fmrA, represses flagellar synthesis and is the first report of a fimbrial-operon-encoded inhibitor of motility in E. coli O157:H7.

Rapid genoserotyping tool for classification of Salmonella serovars.

We have developed a Salmonella genoserotyping array (SGSA) which rapidly generates an antigenic formula consistent with the White-Kauffmann-Le Minor scheme, currently the gold standard for Salmonella serotyping. A set of 287 strains representative of 133 Salmonella serovars was assembled to validate the array and to test the array probes for accuracy, specificity, and reproducibility. Initially, 76 known serovars were utilized to validate the specificity and repeatability of the array probes and their expected probe patterns. The SGSA generated the correct serovar designations for 100% of the known subspecies I serovars tested in the validation panel and an antigenic formula consistent with that of the White-Kauffmann-Le Minor scheme for 97% of all known serovars tested. Once validated, the SGSA was assessed against a blind panel of 100 Salmonella enterica subsp. I samples serotyped using traditional methods. In summary, the SGSA correctly identified all of the blind samples as representing Salmonella and successfully identified 92% of the antigens found within the unknown samples. Antigen- and serovar-specific probes, in combination with a pepT PCR for confirmation of S. enterica subsp. Enteritidis determinations, generated an antigenic formula and/or a serovar designation consistent with the White-Kauffmann-Le Minor scheme for 87% of unknown samples tested with the SGSA. Future experiments are planned to test the specificity of the array probes with other Salmonella serovars to demonstrate the versatility and utility of this array as a public health tool in the identification of Salmonella.

Genetic polymorphisms of innate immunity-related inflammatory pathways and their association with factors related to type 2 diabetes.

BACKGROUND: Type 2 diabetes mellitus (T2DM) has been linked to a state of pre-clinical chronic inflammation resulting from abnormalities in the innate immune pathway. Serum levels of pro-inflammatory cytokines and acute-phase proteins, collectively known as 'inflammatory network', are elevated in the pre-, or early, stages of T2DM and increase with disease progression. Genetic variation can affect the innate immune response to certain environmental factors, and may, therefore, determine an individual's lifetime risk of disease. METHODS: We conducted a cross-sectional study in 6,720 subjects from the Twins UK Registry to evaluate the association between 18 single nucleotide polymorphisms (SNPs) in five genes (TLR4, IL1A, IL6, TNFA, and CRP) along the innate immunity-related inflammatory pathway and biomarkers of predisposition to T2DM [fasting insulin and glucose, HDL- and LDL- cholesterols, triglycerides (TGs), amyloid-A, sensitive C-reactive protein (sCRP) and vitamin D binding protein (VDBP) and body mass index (BMI)]. RESULTS: Of 18 the SNPs examined for their association with nine metabolic phenotypes of interest, six were significantly associated with five metabolic phenotypes (Bonferroni correction, P ≤ 0.0027). Fasting insulin was associated with SNPs in IL6 and TNFA, serum HDL-C with variants of TNFA and CRP and serum sCRP level with SNPs in CRP. Cross-correlation analysis among the different metabolic factors related to risk of T2DM showed several significant associations. For example, BMI was directly correlated with glucose (r = 0.11), insulin (r = 0.15), sCRP (r = 0.23), LDL-C (r = 0.067) and TGs (r = 0.18) but inversely with HDL-C (r = -0.14). sCRP was also positively correlated (P < 0.0001) with insulin (r = 0.17), amyloid-A (r = 0.39), TGs (r = 0.26), and VDBP (r = 0.36) but inversely with HDL-C (r = -0.12). CONCLUSION: Genetic variants in the innate immunity pathway and its related inflammatory cascade is associated with some metabolic risk factors for T2DM; an observation that may provide a rationale for further studying their role as biomarkers for disease early risk prediction.

Plasma vitamin D levels and risk of metabolic syndrome in Canadians.

PURPOSE: Vitamin D deficiency has been implicated in susceptibility to the development of metabolic syndrome, obesity and type 2 diabetes mellitus. The present study aimed to quantify the association between vitamin D plasma level, the number of metabolic syndrome components and insulin resistance in Canadians. METHODS: Vitamin D plasma level and clinical data were determined from 1,818 subjects from the Canadian Health Measures Survey; a representative health survey of the general population of Canada conducted from 2007 to 2009. The definition of metabolic syndrome was based on the National Cholesterol Education Program, Adult Treatment Panel III criteria. Adjusted general linear models were used to estimate the association between vitamin D level and probability of having metabolic syndrome, as well as the association between plasma vitamin D and insulin resistance (homeostasis model assessment for insulin resistance, or HOMA-IR). RESULTS: The prevalence of metabolic syndrome in the study population was 8.9%. The number of metabolic syndrome components was inversely correlated with plasma vitamin D level (ρ= -0.1, p < 0.0001). Subjects in the highest vitamin D quartile had lower odds ratio of metabolic syndrome compared with their counterparts in the lowest vitamin D quartile (0.50, 95% CI= 0.24-1.06). Increasing plasma vitamin D level (by 10 nmol/L) was inversely associated with HOMA-IR score (ß= -0.08, p=0.006) in a model adjusted for physical activity, smoking status, month of interview, age, sex and ethnicity. CONCLUSION: Vitamin D plasma levels are associated with the occurrence of metabolic syndrome components and insulin resistance among Canadians and are linked to increased level of insulin resistance.

Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes.

BACKGROUND: Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. RESULTS: We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. CONCLUSIONS: Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Extending the reach of public health genomics: what should be the agenda for public health in an era of genome-based and "personalized" medicine?

The decade following the completion of the Human Genome Project has been marked by divergent claims about the utility of genomics for improving population health. On the one hand, genomics is viewed as the harbinger of a brave new world in which novel treatments rectify known causes of disease. On the other hand, genomics may have little practical relevance to the principal causes or remedies of diseases which are predominantly social or environmental in origin, particularly in low- and middle-income countries. Those supportive of a role for public health genomics argue that increasing knowledge of genomics and molecular pathology could unlock effective diagnostic techniques and treatments, and better target public health interventions. To resolve some of these tensions, an international multidisciplinary meeting was held in May 2010 in Ickworth, United Kingdom, with the aim of setting an agenda for the development of public health in an era of genome-based and "personalized" medicine. A number of key themes emerged, suggesting a need to reconfigure both the focus for existing genomic research and the stage at which funding is targeted, so that priority is given to areas of greatest potential health impact and that translation from basic science to implementation is given greater emphasis. To support these developments, there should be an immediate, sustained and systematic effort to provide an evidence base. These deliberations formed the basis for six key recommendations, which could guide the practice of public health in an era of genomics and personalized medicine.

Lineage and host source are both correlated with levels of Shiga toxin 2 production by Escherichia coli O157:H7 strains.

Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx(2) flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx(2)) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx(2), whereas all LII strains carried variant stx(2c) and 4 of 14 LI/II strains had copies of both stx(2) and variant stx(2c). Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx(2) mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

In silico genomic analyses reveal three distinct lineages of Escherichia coli O157:H7, one of which is associated with hyper-virulence.

BACKGROUND: Many approaches have been used to study the evolution, population structure and genetic diversity of Escherichia coli O157:H7; however, observations made with different genotyping systems are not easily relatable to each other. Three genetic lineages of E. coli O157:H7 designated I, II and I/II have been identified using octamer-based genome scanning and microarray comparative genomic hybridization (mCGH). Each lineage contains significant phenotypic differences, with lineage I strains being the most commonly associated with human infections. Similarly, a clade of hyper-virulent O157:H7 strains implicated in the 2006 spinach and lettuce outbreaks has been defined using single-nucleotide polymorphism (SNP) typing. In this study an in silico comparison of six different genotyping approaches was performed on 19 E. coli genome sequences from 17 O157:H7 strains and single O145:NM and K12 MG1655 strains to provide an overall picture of diversity of the E. coli O157:H7 population, and to compare genotyping methods for O157:H7 strains. RESULTS: In silico determination of lineage, Shiga-toxin bacteriophage integration site, comparative genomic fingerprint, mCGH profile, novel region distribution profile, SNP type and multi-locus variable number tandem repeat analysis type was performed and a supernetwork based on the combination of these methods was produced. This supernetwork showed three distinct clusters of strains that were O157:H7 lineage-specific, with the SNP-based hyper-virulent clade 8 synonymous with O157:H7 lineage I/II. Lineage I/II/clade 8 strains clustered closest on the supernetwork to E. coli K12 and E. coli O55:H7, O145:NM and sorbitol-fermenting O157 strains. CONCLUSION: The results of this study highlight the similarities in relationships derived from multi-locus genome sampling methods and suggest a "common genotyping language" may be devised for population genetics and epidemiological studies. Future genotyping methods should provide data that can be stored centrally and accessed locally in an easily transferable, informative and extensible format based on comparative genomic analyses.

Host and pathogen determinants of verocytotoxin-producing Escherichia coli-associated hemolytic uremic syndrome.

Verocytotoxin (VT)-producing Escherichia coli (VTEC) infection is associated with a spectrum of clinical manifestations that includes diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome (HUS). The occurrence of HUS in a minority of individuals in outbreaks of VTEC infection is a function of several pathogen and host factors. Pathogen factors include the inoculum size and serotype of the infecting strain, horizontally acquired genetic elements known as pathogenicity islands, and probably the VT type. Host factors that increase the risk of developing HUS include age, pre-existing immunity, gastric acidity, the use of antibiotics and anti-motility agents, and, probably, stress and genetic factors that modulate host response to infection, such as innate immunity and toxin receptor type, expression, and distribution. A better understanding of the pathogen and host determinants of HUS can aid in the development of more effective public health strategies to reduce the risk of developing HUS.

Genomic O island 122, locus for enterocyte effacement, and the evolution of virulent verocytotoxin-producing Escherichia coli.

The locus of enterocyte effacement (LEE) and genomic O island 122 (OI-122) are pathogenicity islands in verocytotoxin-producing Escherichia coli (VTEC) serotypes that are associated with outbreaks and serious disease. Composed of three modules, OI-122 may occur as "complete" (with all three modules) or "incomplete" (with one or two modules) in different strains. OI-122 encodes two non-LEE effector (Nle) molecules that are secreted by the LEE type III secretion system, and LEE and OI-122 are cointegrated in some VTEC strains. Thus, they are functionally linked, but little is known about the patterns of acquisition of these codependent islands. To examine this, we conducted a population genetics analysis, using multilocus sequence typing (MLST), with 72 VTEC strains (classified into seropathotypes A to E) and superimposed on the results the LEE and OI-122 contents of these organisms. The wide distribution of LEE and OI-122 modules among MLST clonal groups corroborates the hypothesis that there has been lateral transfer of both pathogenicity islands. Sequence analysis of a pagC-like gene in OI-122 module 1 also revealed two nonsynonymous single-nucleotide polymorphisms that could help discriminate a subset of seropathotype C strains and determine the presence of the LEE. A nonsense mutation was found in this gene in five less virulent strains, consistent with a decaying or inactive gene. The modular nature of OI-122 could be explained by the acquisition of modules by lateral transfer, either singly or as a group, and by degeneration of genes within modules. Correlations between clonal group, seropathotype, and LEE and OI-122 content provide insight into the role of genomic islands in VTEC evolution.

Molecular analysis as an aid to assess the public health risk of non-O157 Shiga toxin-producing Escherichia coli strains.

Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle with high potential for environmental and zoonotic transmission to humans. Although O157:H7 is the most common STEC serotype, there is growing concern over the emergence of more than 200 highly virulent non-O157 STEC serotypes that are globally distributed, several of which are associated with outbreaks and/or severe human illness such as hemolytic-uremic syndrome (HUS) and hemorrhagic colitis. At present, the underlying genetic basis of virulence potential in non-O157 STEC is unknown, although horizontal gene transfer and the acquisition of new pathogenicity islands are an expected origin. We used seropathotype classification as a framework to identify genetic elements that distinguish non-O157 STEC strains posing a serious risk to humans from STEC strains that are not associated with severe and epidemic disease. We report the identification of three genomic islands encoding non-LEE effector (nle) genes and 14 individual nle genes in non-O157 STEC strains that correlate independently with outbreak and HUS potential in humans. The implications for transmissible zoonotic spread and public health are discussed. These results and methods offer a molecular risk assessment strategy to rapidly recognize and respond to non-O157 STEC strains from environmental and animal sources that might pose serious public health risks to humans.

Applicability of phylogenetic methods for characterizing the public health significance of verocytotoxin-producing Escherichia coli strains.

Two phylogenetic methods (multilocus sequence typing [MLST] and a multiplex PCR) were investigated to determine whether phylogenetic classification of verocytotoxin-producing Escherichia coli serotypes correlates with their classification into groups (seropathotypes A to E) based on their relative incidence in human disease and on their association with outbreaks and serious complications. MLST was able to separate 96% of seropathotype D and E serotypes from those that cause serious disease (seropathotypes A to C), whereas the multiplex PCR lacked this level of seropathotype discrimination.

Citrobacter rodentium virulence in mice associates with bacterial load and the type III effector NleE.

Severe disease caused by Shiga toxin-producing Escherichia coli (STEC) has been associated with a pathogenicity island, O-Island 122, which encodes the type III secretion system-effector NleE. Here we show that full virulence of the related attaching and effacing mouse pathogen Citrobacter rodentium requires NleE. Relative to wild-type bacteria, nleE-mutant C. rodentium are attenuated for colonisation in mice in both single and mixed infections. Examination of the ability of nleE-mutant bacteria to induce pathologic change in vivo revealed that nleE-mutant bacteria induce significantly less pathologic change than wild-type bacteria in susceptible mice. Consistent with these results, mice infected with nleE-mutant bacteria exhibit delayed mortality. These results suggested that pathologic change during attaching and effacing pathogen infection may associate with the degree of pathogen colonisation. Using mutants of 23 type III secretion genes, including the type III effectors nleC, nleD, nleE and nleF, the association of pathologic change with the ability of these mutants to colonise mice was examined. The induction of in vivo disease correlates strongly with the degree of colonisation, suggesting that the colonisation advantage type III secretion genes afford the bacteria, contribute to, and are required for, full virulence.

Use of comparative genomics as a tool to assess the clinical and public health significance of emerging Shiga toxin-producing Escherichia coli serotypes.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause sporadic or epidemic food- or water-borne illness whose clinical spectrum includes diarrhea, hemorrhagic colitis, and the potentially fatal hemolytic uremic syndrome (HUS). Over 200 STEC serotypes have now been implicated in human disease. Serotype O157:H7 is associated most outbreaks and most cases of HUS. Other serotypes are also associated with outbreaks and HUS but less commonly than serotype O157:H7, and some cause HUS but are typically non-epidemic. Many STEC serotypes have been associated with diarrhea, but not with outbreaks or HUS, while others, isolated from cattle, have never been linked to human disease. The only proven virulence strategies for STEC are Stx production and, in some strains, a characteristic attaching and effacing cytopathology on enterocyte that is mediated by factors encoded on a pathogenicity island (PAI) known as the locus of enterocyte effacement (LEE). But Stx subtypes and LEE cannot fully explain the apparent differences in virulence between STEC subgroups. However, publication of the genome sequences of two E. coli O157:H7 strains has revealed new candidate PAIs and has stimulated the use of novel approaches for assessing differences in virulence potential between groups of strains. This paper highlights the clinico-pathological features and pathogenesis of STEC infection, new information arising from E. coli O157:H7 genome sequences, and progress in the use of of comparative genomics for assessing potential differences in virulence and public health significance between STEC subgroups.