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.

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.

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.

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.

Methodologies towards the development of an oligonucleotide microarray for determination of Salmonella serotypes.

A DNA-based microarray designed to detect somatic (O) and flagellar (H) antigens present in the five most commonly isolated Salmonella serovars within Canada was developed as an alternative to the traditional Kauffmann-White serotyping scheme currently used to serotype salmonellae. Short oligonucleotide probes were designed based on publicly available sequence data of selected genes responsible for O and H antigen biosynthesis. These targets included: antigen-specific sequences within the flagella (H) antigen phase 1 (fliC) and phase 2 (fljB) genes and somatic (O) antigen biosynthesis genes within the rfb cluster (Groups B--rfbJ, C1--wbaA, C2--rfbJ, D1--rfbS). A prototype microarray with 117 O and H antigen-specific probes and controls was used to assess probe performance against two pools of gene target PCR amplicons. A set of 31 of these antigen-specific probes (8 O and 23 H) with high specific signal and low non-specific signal were selected based on t-test (p-value <0.01) and log(2) ratio distribution analysis to create a prototype microarray. The microarray was tested against 16 Salmonella strains of known serotype. Based on the strains tested in this study, these probes successfully identified and differentiated 11 of the 12 antigens targeted. The prototype DNA-based typing microarray described here has the potential to be an automated alternative to the traditional antigen-antibody serotyping scheme currently used for Salmonella.

Identification of Escherichia coli O157:H7 genomic regions conserved in strains with a genotype associated with human infection.

Beta-glucuronidase-negative, sorbitol-nonfermenting isolates of Shiga toxin-producing Escherichia coli O157 comprise part of a clone complex of related enterohemorrhagic E. coli isolates. High-resolution genotyping shows that the O157 populations have diverged into two different lineages that appear to have different ecologies. To identify genomic regions unique to the most common human-associated genotype, suppression subtractive hybridization was used to identify DNA sequences present in two clinical strains representing the human lineage I O157:H7 strains but absent from two bovine-derived lineage II strains. PCR assays were then used to test for the presence of these regions in 10 lineage I strains and 20 lineage II strains. Twelve conserved regions of genomic difference for lineage I (CRD(I)) were identified that were each present in at least seven of the lineage I strains but absent in most of the lineage II strains tested. The boundaries of the lineage I conserved regions were further delimited by PCR. Eleven of these CRD(I) were associated with E. coli Sakai S-loops 14, 16, 69, 72, 78, 82, 83, 91 to 93, 153, and 286, and the final CRD(I) was located on the pO157 virulence plasmid. Several potential virulence factors were identified within these regions, including a putative hemolysin-activating protein, an iron transport system, and several possible regulatory genes. Cluster analysis based on lineage I conserved regions showed that the presence/absence of these regions was congruent with the inferred phylogeny of the strains.

Salmonella phages and prophages--genomics and practical aspects.

Numerous bacteriophages specific to Salmonella have been isolated or identified as part of host genome sequencing projects. Phylogenetic analysis of the sequenced phages, based on related protein content using CoreGenes, reveals that these viruses fall into five groupings (P27-like, P2-like, lambdoid, P22-like, and T7-like) and three outliers (epsilon15, KS7, and Felix O1). The P27 group is only represented by ST64B; the P2 group contains Fels-2, SopEphi, and PSP3; the lambdoid Salmonella phages include Gifsy-1, Gifsy-2, and Fels-1. The P22-like viruses include epsilon34, ES18, P22, ST104, and ST64T. The only member of the T7-like group is SP6. The properties of each of these phages are discussed, along with their role as agents of genetic exchange and as therapeutic agents and their involvement in phage typing.