Shiga toxin 2 overexpression in Escherichia coli O157:H7 strains associated with severe human disease

Variation in disease severity among Escherichia coli O157:H7 infections may result from differential expression of Shiga toxin 2 (Stx2). Eleven strains belonging to four prominent phylogenetic clades, including clade 8 strains representative of the 2006 U.S. spinach outbreak, were examined for stx2 expression by real-time PCR and western blot analysis. Clade 8 strains were shown to overexpress stx2 basally, and following induction with ciprofloxacin when compared to strains from clades 1-3. Differences in stx2 expression generally correlated with Stx2 protein levels. Single-nucleotide polymorphisms identified in regions upstream of stx2AB in clade 8 strains were largely absent in non-clade 8 strains. This study concludes that stx2 overexpression is common to strains from clade 8 associated with hemolytic uremic syndrome, and describes SNPs which may affect stx2 expression and which could be useful in the genetic differentiation of highly-virulent strains.Microbiology and Molecular Genetic

Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

<p>Abstract</p> <p>Background:</p> <p>Global patterns of gene expression of <it>Escherichia coli </it>K-12 during growth transitions have been deeply investigated, however, comparable studies of <it>E. coli </it>O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were confirmed by Q-PCR.</p> <p>Results:</p> <p>Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (<it>n </it>= 1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold. Most ORFs of the LEE pathogenicity island also decreased ~2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as <it>tccP </it>and <it>espJ</it>, also decreased in expression from exponential to stationary phase. Three of the clusters (<it>n </it>= 154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (<it>n </it>= 923) which includes genes involved in stress responses (e.g. <it>gadAB</it>, <it>osmBC</it>, and <it>dps</it>). These transcript levels remained relatively high for > 3 h in stationary phase. The Shiga toxin genes (<it>stx</it>1AB and <it>stx</it>2B) were significantly induced after transition into stationary phase.</p> <p>Conclusion:</p> <p>Expression of more than 300 O157-specific ORFs, many implicated in virulence of the O157 pathogen, was modulated in a growth dependent manner. These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions.</p

Sigma factor n, liaison to an ntrC and rpoS dependent regulatory pathway controlling acid resistance and the LEE in enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σ(N)), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σ(N)-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σ(S)) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σ(S) was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σ(N) allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σ(S) stability, gadE, ler or GDAR, indicating a dependence on transcription from a σ(N) promoter(s) and not RNAP competition for the phenotype. Among a library of σ(N) enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σ(S) stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σ(N) regulate GDAR and LEE expression through downregulation of σ(S) at the post-translational level; likely by altering σ(S) stability or activity. The regulatory interplay between NtrC, other EBPs, and σ(N)-σ(S), represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.Microbiology and Molecular Genetic

Increased Adherence and Expression of Virulence Genes in a Lineage of Escherichia coli O157:H7 Commonly Associated with Human Infections

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a food and waterborne pathogen, can be classified into nine phylogenetically distinct lineages, as determined by single nucleotide polymorphism genotyping. One lineage (clade 8) was found to be associated with hemolytic uremic syndrome (HUS), which can lead to kidney failure and death in some cases, particularly young children. Another lineage (clade 2) differs considerably in gene content and is phylogenetically distinct from clade 8, but caused significantly fewer cases of HUS in a prior study. Little is known, however, about how these two lineages vary with regard to phenotypic traits important for disease pathogenesis and in the expression of shared virulence genes.Here, we quantified the level of adherence to and invasion of MAC-T bovine epithelial cells, and examined the transcriptomes of 24 EHEC O157:H7 strains with varying Shiga toxin profiles from two common lineages. Adherence to epithelial cells was >2-fold higher for EHEC O157:H7 strains belonging to clade 8 versus clade 2, while no difference in invasiveness was observed between the two lineages. Whole-genome 70-mer oligo microarrays, which probe for 6088 genes from O157:H7 Sakai, O157:H7 EDL 933, pO157, and K12 MG1655, detected significant differential expression between clades in 604 genes following co-incubation with epithelial cells for 30 min; 186 of the 604 genes had a >1.5 fold change difference. Relative to clade 2, clade 8 strains showed upregulation of major virulence genes, including 29 of the 41 locus of enterocyte effacement (LEE) pathogenicity island genes, which are critical for adherence, as well as Shiga toxin genes and pO157 plasmid-encoded virulence genes. Differences in expression of 16 genes that encode colonization factors, toxins, and regulators were confirmed by qRT-PCR, which revealed a greater magnitude of change than microarrays.These findings demonstrate that the EHEC O157:H7 lineage associated with HUS expresses higher levels of virulence genes and has an enhanced ability to attach to epithelial cells relative to another common lineage

Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac

<p>Abstract</p> <p>Background</p> <p>Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in <it>S. aureus </it>strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (Dc^RS) <it>S. aureus </it>strains.</p> <p>Methods</p> <p>Transcriptional alterations in response to growth with diclofenac were measured using <it>S. aureus </it>gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry.</p> <p>Results</p> <p>Growth of <it>S. aureus </it>strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including <it>mepRAB </it>and a putative <it>emrAB/qacA</it>-family pump. Diclofenac up-regulated <it>sigB </it>(σ^B), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. <it>Staphylococcus aureus </it>microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σ^B-regulated. Diclofenac altered <it>S. aureus </it>susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to Dc^RSdid not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation.</p> <p>Conclusions</p> <p>The results of this study suggest that diclofenac influences antibiotic susceptibility in <it>S. aureus</it>, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.</p

Representations of sport in the revolutionary socialist press in Britain, 1988–2012

This paper considers how sport presents a dualism to those on the far left of the political spectrum. A long-standing, passionate debate has existed on the contradictory role played by sport, polarised between those who reject it as a bourgeois capitalist plague and those who argue for its reclamation and reformation. A case study is offered of a political party that has consistently used revolutionary Marxism as the basis for its activity and how this party, the largest in Britain, addresses sport in its publications. The study draws on empirical data to illustrate this debate by reporting findings from three socialist publications. When sport did feature it was often in relation to high profile sporting events with a critical tone adopted and typically focused on issues of commodification, exploitation and alienation of athletes and supporters. However, readers’ letters, printed in the same publications, revealed how this interpretation was not universally accepted, thus illustrating the contradictory nature of sport for those on the far left

Alterations In the Transciptome and Antibiotic Susceptibility of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e Grown In the Presence of Diclofenac

Background Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. Methods Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Results Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. Conclusions The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport

Intravenous ascorbic acid to prevent and treat cancer-associated sepsis?

The history of ascorbic acid (AA) and cancer has been marked with controversy. Clinical studies evaluating AA in cancer outcome continue to the present day. However, the wealth of data suggesting that AA may be highly beneficial in addressing cancer-associated inflammation, particularly progression to systemic inflammatory response syndrome (SIRS) and multi organ failure (MOF), has been largely overlooked. Patients with advanced cancer are generally deficient in AA. Once these patients develop septic symptoms, a further decrease in ascorbic acid levels occurs. Given the known role of ascorbate in: a) maintaining endothelial and suppression of inflammatory markers; b) protection from sepsis in animal models; and c) direct antineoplastic effects, we propose the use of ascorbate as an adjuvant to existing modalities in the treatment and prevention of cancer-associated sepsis

A multi-level analysis of team climate and interpersonal exchange relationships at work

This paper seeks to advance research on interpersonal exchange relationships between supervisors, subordinates, and coworkers at work by integrating social exchange, workplace friendship, and climate research to develop a multi-level model. We tested the model using hierarchical linear modeling (HLM) with data obtained from a sample of 215 manager–employee dyads working in 36 teams. At the individual level, leader–member exchange (LMX) was found to be related to workplace friendship. Further, workplace friendship was positively related to team–member exchange (TMX) and mediated the LMX–TMX relationship. At the team level, HLM results indicated that the relationship between LMX and workplace friendship was moderated by affective climate. These findings suggest that high-quality LMX relationships are associated with enhanced workplace friendship between employees, especially when the affective climate is strong

A survey of security issue in multi-agent systems

Multi-agent systems have attracted the attention of researchers because of agents' automatic, pro-active, and dynamic problem solving behaviors. Consequently, there has been a rapid development in agent technology which has enabled us to provide or receive useful and convenient services in a variety of areas such as banking, transportation, e-business, and healthcare. In many of these services, it is, however, necessary that security is guaranteed. Unless we guarantee the security services based on agent-based systems, these services will face significant deployment problems. In this paper, we survey existing work related to security in multi-agent systems, especially focused on access control and trust/reputation, and then present our analyses. We also present existing problems and discuss future research challenges. © Springer Science+Business Media B.V 2011